(Received for publication, July 21, 1995; and in revised form, October 7, 1995)
From the
The phosphorylation of cardiac muscle troponin I (CTnI) at two
adjacent N-terminal serine residues by cAMP-dependent protein kinase
(PKA) has been implicated in the inotropic response of the heart to
-agonists. Phosphorylation of these residues has been shown to
reduce the Ca
affinity of the single
Ca
-specific regulatory site of cardiac troponin C
(CTnC) and to increase the rate of Ca
dissociation
from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J.,
Kranias, E. G., Potter, J. D., and Solaro, R. J.(1982) J. Biol.
Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J.,
and Potter, J. D.(1995) Circ. Res. 76, 1028-1035) have
correlated this increase in Ca
dissociation with a
reduced Ca
sensitivity of force development and a
faster rate of cardiac muscle relaxation in a PKA phosphorylated
skinned cardiac muscle preparation. To further determine the role of
the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23),
serine 22 or 23, or both were mutated to alanine. The wild type and the
mutated CTnIs were expressed in Escherichia coli and purified.
Using these mutants, it was found that serine 23 was phosphorylated
more rapidly than serine 22 and that both serines are required to be
phosphorylated in order to observe the characteristic reduction in the
Ca
sensitivity of force development seen in a skinned
cardiac muscle preparation. The latter result confirms that PKA
phosphorylation of CTnI, and not other proteins, is responsible for
this change in Ca
sensitivity. The results also
suggest that one of the serines (23) may be constitutively
phosphorylated and that serine 22 may be functionally more important.
Several lines of evidence have led to a general understanding of
how -agonist stimulation leads to positive inotropic and
chronotropic effects by phosphorylation of cellular substrates through
cAMP-dependent protein kinase
(PKA)(
)(1, 2) . It is generally agreed that
phosphorylation of sarcolemmal Ca
channels (3) and phospholamban(4, 5) , a sarcoplasmic
reticular protein regulating the Ca
pump, are
responsible for the changes seen in the intracellular Ca
transient. The resulting increases in intracellular
Ca
and the rate of Ca
resequestration contribute to an increase in cardiac muscle
contractility and to faster rates of force development and relaxation.
PKA can also phosphorylate contractile machinery proteins, such as
C-protein and cardiac troponin I (CTnI) (6, 7, 8, 9) . Many laboratories,
including ours, have focused on the mechanism by which phosphorylation
of CTnI may be involved in the inotropic effect brought about by
-agonist stimulation.
Troponin I (TnI), a subunit of the
troponin complex, inhibits the actomyosin ATPase activity when muscle
is in the resting state. Binding of Ca to the low
affinity site(s) of troponin C (TnC) releases the TnI inhibition on
actomyosin ATPase through protein-protein interactions among the
troponin complex, tropomyosin and actin, and leads to muscle
contraction(10) . In comparison with skeletal TnI, CTnI has an
additional 32-33 amino acids at its N terminus, and this segment
also contains two adjacent serine residues at positions 22 and 23 or 23
and 24, depending on the
species(11, 12, 13) . By converting the
phosphoserine into the stable S-ethylcysteine derivative on
bovine CTnI, Swiderek et al.(14, 15) demonstrated that these two serines were
able to be fully phosphorylated by PKA. Biochemical studies have shown
that CTnI can be phosphorylated to the level of 2 mol of phosphate/mol
of protein (16) , consistent with the sequence prediction.
Considerable effort has been made with either perfused hearts (17, 18) or isolated proteins (14, 15, 16) to determine the mechanism by
which CTnI phosphorylation may affect cardiac muscle contraction. The
decreased Ca
sensitivity upon phosphorylation of CTnI
has been observed in isolated cardiac
myofibrils(8, 19) , in hyperpermeable cardiac
fibers(20) , and in skinned cardiac muscle preparations and
myocytes(21, 22) . Moreover, it has been shown that
CTnI phosphorylation increases the rate of Ca
dissociation from a reconstituted troponin complex(23) ,
suggesting the possible importance of this phosphorylation in
modulating the rate of relaxation of cardiac muscle. Some reports,
however, have shown that CTnI remains phosphorylated even after removal
of
-agonists and the return of muscle contraction to prestimulated
basal levels(4, 19) . These results have suggested
that phosphorylation of CTnI may not be as important as the
phosphorylation of other proteins, such as phospholamban(4) .
In a recent study we have clearly shown that CTnI phosphorylation is
directly correlated with the faster muscle relaxation seen after PKA
phosphorylation and is probably due to the decreased Ca
affinity of CTnC and the consequent faster dissociation of
Ca
(22) . Our calculations also showed that
although PKA phosphorylation of phospholamban is the predominant
effector in the inotropic response, the rate of relaxation is
significantly influenced by CTnI phosphorylation.
After many years
of study there are still numerous questions regarding the mechanism of
action of CTnI phosphorylation and of the physiological significance of
this process. In the present study we have focussed on the role of the
phosphorylation of the two CTnI serines in this process. In order to
study the functional and physiological significance of phosphorylation
of these two serine residues, we have used our cloned mouse CTnI cDNA
to create three mutants of CTnI in which either serine 22 or 23, or
both, are mutated into alanine(9) . Our results demonstrate
that both serine residues in CTnI are able to be phosphorylated by PKA in vitro but that the rates of phosphorylation for these two
serine residues are different. Using a reconstituted skinned cardiac
muscle preparation (CSM), we have studied the effect of phosphorylation
of each serine residue on the Ca dependence of force
development. We show that phosphorylation of both serine residues by
PKA is required for the decreased Ca
-sensitivity of
force development. Since the rate of phosphorylation is different, it
is possible that the rate of dephosphorylation is different and may
account for the slower dephosphorylation seen in intact systems (4) and suggests that one of the serines may be more or less
constitutively phosphorylated with the other serine being functionally
more important.
Skeletal muscle was isolated from rabbit psoas muscle and immersed for 30 min in pCa 8.0 relaxation solution at 4 °C. Then the muscle bundles was excised into small bundles (2-4 mm in diameter, 8-10 cm in length) and tied to wooden sticks. The bundles were incubated in pCa 8.0 solution containing 50% glycerol for overnight at 4 °C. The bundles were changed to a fresh pCa 8.0 solution containing 50% glycerol and then stored at -20 °C until use.
Figure 1:
SDS-PAGE of bacterially
synthesized wild type CTnI and its mutants. A: lane
1, bovine cardiac CTnI; lane 2, bacterial lysate with
CTnI expression; lane 3, bacteria lysate without CTnI
expression. B, purified wild type and mutant CTnIs. Lane 1,
purified wild type CTnI; lane 2, purified CTnI; lane 3, purified CTnI
; lane 4,
purified CTnI
; lane 5, bovine
CTnI.
Figure 2:
The
time courses of phosphorylation of wild type CTnI and its mutants.
Phosphorylation of wild type CTnI and its mutants was performed as
described under ``Materials and Methods.'' A-C, autoradiograms showing the phosphorylation of wild
type and mutant CTnIs. 1 µg of either wild type CTnI or mutants
were phosphorylated by PKA under conditions described under
``Materials and Methods.'' The phosphorylation reactions were
stopped with the SDS sample buffer added to the reaction mixtures at
different reaction times. Portions of these samples were
electrophoresed on 15% SDS-PAGE, and the dried gels were exposed to
x-ray films. A: lanes 1-8, phosphorylation of wild type
CTnI; B: lanes 1-8, phosphorylation of
CTnI; C: lanes 1-8,
phosphorylation of CTnI
. Lanes 1-8 in the
figures represent the different times of phosphorylation. They are 30
s, 1 min, 2 min, 5 min, 10 min, 30 min, and 60 min, respectively. D, time course of phosphate incorporation into wild type and
mutant CTnIs by PKA. The amount of phosphorylation of the wild type and
mutant CTnIs was calculated as described under ``Materials and
Methods'' and was plotted as an average of three to four
experiments (X = mean ±
S.E.).
Figure 3:
Effect of PKA on the Ca sensitivity of muscle contraction. The Ca
sensitivity of muscle contraction was determined as described
under ``Materials and Methods.'' Then the CSM was incubated
in the phosphorylation solution with PKA for 1 h or without PKA for
1-2 h. The Ca
sensitivity of muscle contraction
after incubation was determined again. Solid circle with solid
line, Ca
sensitivity of muscle contraction
before incubation; solid inverted triangle with long dashed
line, Ca
sensitivity of muscle contraction after
incubation with PKA; solid triangle with short dashed line,
Ca
sensitivity of muscle contraction after incubation
with phosphorylation solution without PKA. Inset: left, autoradiogram of the SDS gel (on right) of
phosphorylated CSM.
Figure 4:
CTnI extraction and reconstitution. CSM
was tested for its initial force and was then extracted by treatment
with the 10 mM orthovanadate solution described under
``Materials and Methods'' for 10 min. After wash out of the
vanadate with the pCa 8.0 solution, the CSM lost its
Ca dependence due to the loss of CTnI and CTnC. When
maximal force was obtained, the muscle was incubated with 10 µM CTnI
CTnC complex in pCa 8.0 solution. The force
restoration was tested after a 3-h incubation in CTnI
CTnC
complex. The scales of different time blocks during the experiment are
indicated in the figures. The total time of time block II in A and B is 10 min. A, the CTnI
CTnC complex
was used for reconstitution; B, control experiment. No
CTnI
CTnC was used for the muscle reconstitution. C, Coommasie Blue-stained SDS-PAGE of solubilized CSM which has been
extracted by vanadate treatment and reconstituted with CTnI
CTnC
complex. Lane 1, bovine CTnI; lane 2, nonextracted
CSM; lane 3, extracted CSM; lane 4, same as lane
3, but the load as twice as much; lane 5, reconstituted
CSM.
The amount of CTnI
extraction and reconstitution was further checked by SDS-PAGE as
demonstrated in Fig. 4C. After vanadate treatment, more
than 90% of the endogenous CTnI was extracted. The relative content of
other proteins after vanadate treatment was analyzed using the
Gelbase/Gelblot program (Ultra Violet Product, version 1.96, 1995).
Although the quantities of the proteins loaded onto the gel shown in Fig. 4C, before and after extraction and after
reconstitution, were different, the relative amount of protein before
and after extraction remained the same (Table 1), suggesting that
vanadate specifically extracted CTnI and CTnC without significantly
affecting other proteins. The incubation of the extracted CSM with the
CTnICTnC complex resulted in a relatively complete CTnI
reconstitution (Table 1). These results are consistent with the
findings described in Fig. 4, A and B, i.e. the loss of Ca
-dependent force after
the vanadate treatment and the restoration of
Ca
-dependent force of the CSM following its
reconstitution with the CTnI
CTnC complex. Unfortunately, it was
not possible to quantify the extraction and reconstitution of CTnC in
CSM due to its poor staining properties (28) .
Figure 5:
Phosphorylation of wild type CTnI and its
mutants after they were reconstituted back into CTnI-depleted CSM. The
photographs represent a Coomassie Blue-stained gel and autoradiogram of
the same gel of the solubilized CSM phosphorylated by PKA. The
extraction of endogenous CTnI from CSM and the reconstitution of wild
type CTnI and its mutants were the same as described in the legend to Fig. 4. Phosphorylation of the reconstituted CSM was described
under ``Materials and Methods.'' Lanes 1-4 are
an SDS-PAGE representing CSM reconstituted with wild type CTnI,
CTnI, CTnI
, and CTnI
,
respectively. Lanes 5-8 are the autoradiography of lanes 1-4.
Fig. 6illustrates the
changes in the Ca dependence of force development
brought about by PKA phosphorylation. Interestingly, the only
PKA-induced change in the Ca
dependence of force
development was seen in the CSM reconstituted with wild type CTnI.
Phosphorylation of this preparation resulted in a decrease in the pCa
by
0.18 pCa units. No change in
the Ca
dependence of force development was observed
after phosphorylation of the CSM reconstituted with any of the three
mutated CTnIs, independent of the location of the mutation (Table 2). These results show that phosphorylation of both Ser-22
and Ser-23 are required to observe the change in the Ca
dependence of force development and confirm that PKA
phosphorylation of CTnI, and not other proteins, is responsible for the
reduction in the Ca
sensitivity of force development.
There is no correlation between the extent of recovery of force and the
observed pCa
of force development. However, the pCa
of the reconstituted CSM was 0.17-0.28 pCa units higher than the unextracted CSM (Table 2).
There are several possibilities that may explain this. 1) The CTnI in
the unextracted CSM was already partially phosphorylated, producing a
lower Ca
sensitivity. This does not seem possible,
since the pCa
of the phosphorylated reconstituted
CSM was higher than that of phosphorylated unextracted CSM. Also
contradictory was the data demonstrating that partially phosphorylated
CTnI had no effect on the Ca
sensitivity of muscle
contraction. 2) Even after 3 h incubation with CTnI
CTnC in the pCa 8.0 solution, there was still some
Ca
-independent force, presumably caused by
cross-bridge attachment due to incomplete CTnI
CTnC
reconstitution. Therefore, the attached cross-bridges may be
responsible for the increased Ca
sensitivity seen in
the reconstituted CSM(26) .
Figure 6:
Effect of PKA on the Ca dependence of force development of extracted CSM subsequently
reconstituted with the complex of CTnC and either wild type or mutated
CTnIs. The extraction and reconstitution procedures and the force
measurements were the same as described in the legend to Fig. 4.
The results are all based on comparison of the same CSM before and
after treatment with PKA. Each CSM was its own control. The results are
an average of five to seven independent determinations, and the
statistical analysis is shown in Table 2. Phosphorylation was
performed as described under ``Materials and Methods.'' The
CTnC
CTnI complexes used for reconstitution were formed utilizing
bovine CTnC and wild type CTnI: A, CTnI
; B, CTnI
; C, CTnI
; D, CTnI
.
As we have previously reported(9) , mouse CTnI can be expressed in a bacterial expression system. The purification method presented here has proven to be an efficient way to obtain large quantities of CTnI (30) . However, the final yield of CTnI purified from the pET expression system is not as high as for skeletal TnI(29) . This probably results from the codon usages for the first 32 amino acids in the mouse CTnI cDNA sequence, since the expression of a deletion mutant of CTnI devoid of the first 32 amino acids was much higher than that of the intact CTnI(30) .
PKA
phosphorylation of CTnI causes a decrease in the Ca dependence of force development or of the myofibrillar ATPase of
cardiac muscle and has been observed by many
investigators(8, 9, 20, 21, 36) .
We have recently found that CTnI phosphorylation increases the rate of
Ca
dissociation from CTnC, thus contributing to the
observed faster relaxation(22, 23) . CTnI contains two
adjacent serine residues immediately adjacent to three arginine
residues, i.e. R-R-R-S-S, which meets the minimal sequence
requirement (R-R-X-S/T) for PKA phosphorylation of both
serines(31) . Various studies on CTnI phosphorylation have
suggested that CTnI can be phosphorylated up to 2 mol of phosphate/mol
of protein(15, 16, 32) , which is consistent
with the amino acid sequence of the CTnIs. Our phosphorylation studies
have confirmed the prediction that these two serine residues are able
to be phosphorylated by PKA. Moreover we have shown that these two
serines are the only phosphorylation sites in CTnI, since the mutant,
CTnI
was not phosphorylated by PKA. In agreement
with the study by Mittmann et al.(32) , we found that
the rate constants for the phosphorylation of the two serines are
different, with Ser-22 being phosphorylated at a slower rate than
Ser-23. However, in contrast to the peptide studies (32) , the
difference between the rates of phosphorylation of the two serines was
not 13-fold but more like 2-fold. The reason of the observed
discrepancy is probably due to the different conformation of the two
substrates used for phosphorylation. In contrast to our studies on
whole CTnI, the peptide used by Mittmann et al. (11) was only ten amino acids in length. The other interesting
finding is that the phosphorylation of the wild type CTnI was equal to
the sum of the two mutant CTnI phosphorylation. This implies that the
phosphorylation of Ser-22 and Ser-23 are independent of each other.
We have demonstrated that only the phosphorylated wild type CTnI was
effective in decreasing the Ca sensitivity of force
development. The three other mutants, when substituted into CSM, showed
no effect of PKA on the Ca
dependence of force
development. The in vitro experiments ( Fig. 2and Fig. 5) have demonstrated that both CTnI
and
CTnI
can be fully phosphorylated. Our results suggest
then that both serines need to be phosphorylated to lower the
Ca
sensitivity of muscle contraction. The
monophosphate forms of CTnI do not appear to be effective in decreasing
the Ca
sensitivity of muscle contraction.
It is of
course possible that, due to the limitation of the methodology used, we
were unable to observe small effects when only Ser-22 or Ser-23 are
phosphorylated. The limitation of using the vanadate extraction and
reconstitution method is that it is difficult to restore the
Ca-dependent force close to the initial force before
extraction. This is in part due to the fall off in force that occurs
during the incubation of the extracted fiber with the CTnI
CTnC
complex (Fig. 4, A and B). It may also explain
why the change in the Ca
dependence of force
development in CSM reconstituted with wild type CTnI brought about by
PKA is only
0.18 pCa units versus a change of
0.26 pCa observed in nonextracted CSM (Table 1).
Although we cannot rule out the possibility that these intrinsic
methodological weaknesses might contribute to our failure to see any
effect of the single serine phosphorylation on the Ca
dependence, our data are consistent with other many experimental
results. In any event, our results clearly show that PKA
phosphorylation of CTnI, and not some other protein(s), is responsible
for the PKA induced change in Ca
sensitivity.
It
has been reported that the majority of the endogenous phosphate in CTnI
isolated from cardiac tissue is contained in the second serine (14) . It is also possible to isolate the monophosphate form of
native CTnI, in which only the second serine is phosphorylated, from
cardiac tissue(33) . Isolation of the monophosphate form of
CTnI in which only the first serine is phosphorylated has not been
reported, although it appears to exist(33) . Therefore, even in
the resting state when -agonist levels are low, CTnI is probably
partially phosphorylated, mostly at the second serine. These data
suggest that not only are the kinetics of phosphorylation of these two
serine residues different, but more importantly, phosphorylation of
serine 23 alone probably does not seem to affect cardiac muscle
contractility. Second, CTnI isolated from cardiac muscle always
contains different levels of endogenous phosphate, from 0.5 to 1.5 mol
of phosphate/mol of protein(16) . These differences, as pointed
out by Swidirek(14) , are probably due to the different methods
used to measure the absolute content of phosphate. The interesting
point here is that upon
-agonist stimulation, the net increase in
the phosphate of CTnI is
1 mol of phosphate/mol of
protein(16) . Concerning the fact that Ser-23 is phosphorylated
even in the resting state, it is reasonable to assume that the net
increase of phosphate after
-agonist stimulation is mostly added
on the first serine or Ser-22 in the mouse. Thus Ser-23 appears to be
constitutively phosphorylated, with the phosphate of Ser-22 being
functionally more important. This also suggests that the rate of
dephosphorylation of these two serines is probably different and may
account for some of the observations in the in vivo studies(4, 19) . Third, using
P NMR
spectroscopy, Jaquet et al.,(33) found that a new
P NMR signal appeared only when both Ser-23 and Ser-24 of
bovine CTnI (equivalent to Ser-22 and Ser-23 in mouse CTnI) were
phosphorylated in a complex formed with TnT and TnC. Their result
suggests that these two phosphorylated serine residues produce a
specific interaction within the troponin complex, possibly causing
Ca
to dissociate faster from CTnC. The two
monophospho-forms of CTnI did not show any changes in this specific
subunit interaction.
All of these results suggest that partially
phosphorylated CTnI has little or no effect on the Ca dependence of force development and therefore on cardiac
contractility. Only the doubly phosphorylated form of CTnI appears to
contribute to these processes. In combining these results with our
phosphorylation data on wild type CTnI and its mutants, we hypothesize
that this type of ordered phosphorylation may indeed occur in native
CTnI with the major change in phosphorylation occurring on serine 22.
These results raise the question as to what the physiological
significance of the existence of the two adjacent serine residues in
CTnI is and how the phosphorylation of these two serines affects
Ca binding to CTnC. At this point, we do not have a
satisfactory answer. The existing sequence data shows that the
existence of the two adjacent serine residues on CTnI is shared by
avian, and many other mammalian species, implying the possible
physiological necessity of the existence of these two serine residues.
The effect of CTnI phosphorylation on the Ca
affinity
of TnC is achieved most likely through a change in the interaction
between CTnI and CTnC, which probably requires phosphorylation of both
serine residues. Sheng et al. (29) have demonstrated
that the N-terminal domain of skeletal TnI interacts with TnC in a
Ca
-Mg
site-dependent manner, and
this interaction serves to maintain the structure of troponin complex.
Recent NMR studies on the spatial relationships within the
CTnI
CTnC complex demonstrated that CTnI and CTnC also form an
antiparallel arrangement similar to skeletal
TnI(34, 35) . Since the two adjacent serines are close
to the TnC binding domain in the N-terminal portion CTnI, the phosphate
introduced by PKA possibly influences the interaction between the N
terminus of CTnI and the C terminus of CTnC. This structural change may
also affect the Ca
-dependent interaction between the
N-terminal region of CTnC and CTnI. The monophosphate form of CTnI may
play only a transitory role. Further studies on the interaction between
CTnI and CTnC using molecular modeling and their structural
determination will help to define these interactions.
In summary,
our current view of the mechanism by which CTnI phosphorylation
controls the increased rate of muscle relaxation is that: 1) in the
resting state, CTnI is partially phosphorylated, mostly at the second
serine; 2) when PKA is activated by the -agonist pathway, PKA
further phosphorylates TnI, mostly at the first serine; 3) the
Ca
sensitivity of muscle contraction decreases when
both serines on CTnI are phosphorylated by PKA, allowing Ca
to dissociate faster from the single
Ca
-specific regulatory site of CTnC; 4) after removal
of
-agonists, CTnI is dephosphorylated, primarily at the first
serine.