Department of Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109-0622
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.
gene transfer; myocyte; thin filament
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-ADRENERGIC
ACTIVATION of protein kinase A (PKA) in cardiac myocytes reduces
the Ca2+ sensitivity of myofilament tension
(15), and this response is hypothesized to contribute to
accelerated relaxation in the intact heart (42). One
myofilament protein phosphorylated in response to cAMP-dependent PKA
activation is cardiac troponin I (cTnI; Ref. 8), a key
regulatory protein of the thin filament. Recent investigations indicate
that cTnI phosphorylation plays an important role in PKA-mediated
decreases in myofilament Ca2+ sensitivity of tension
(3, 11, 15). The increase in cTnI phosphorylation and
decrease in myofilament Ca2+ sensitivity of tension
typically observed with PKA activation (15) are both
markedly attenuated in myocardium expressing the slow skeletal isoform
of TnI (ssTnI), which lacks the PKA phosphorylation domain (3,
11). The relationship between cTnI phosphorylation and
myocardial function appears to be clinically significant, as there is
accumulating evidence that patients experiencing heart failure also
have altered levels of phosphorylated cTnI (4, 5). This
clinical observation underscores the significance of gaining a better
understanding of the role phosphorylated TnI plays in the myofilament
response to
-adrenergic stimulation. An important step toward this
goal is to investigate the domain(s) within cTnI that are responsible
for PKA-induced decreases in the Ca2+ sensitivity of
myofilament tension.
At present, cTnI amino acid residues phosphorylated by PKA are well known, but the region(s) within TnI that may be important for the ensuing changes in myofilament function are not well understood. The primary residues phosphorylated by PKA are serine residues 23 (Ser-23) and 24 (Ser-24) within rodent (43) and human cTnI (24). These serines reside within a unique 32-amino acid amino terminal cTnI extension that is absent in slow or fast skeletal TnI isoforms (19). Al-Hillawi and colleagues (1) proposed that phosphorylation of the amino terminal extension of cTnI by PKA leads directly to changes in the interactions between this region of TnI and other myofilament proteins. Spectroscopic analysis of TnI interactions with TnC supports these hypotheses (12). In contrast, results from fluorescence emission studies indicate that PKA phosphorylation of the amino terminal extension of cTnI may be transmitted through long-range interactions to the carboxyl region of the protein (6). Signal transmission through the carboxyl terminus may be expected because this region influences the Ca2+ sensitivity of myofilament tension (35, 36). In biochemical studies, this region acts as a molecular switch that toggles from actin to troponin C with increasing Ca2+ levels (see Ref. 30 for review). Thus phosphorylation of the amino terminal extension of cTnI could affect this isoform-specific, molecular switch domain as a means of influencing myofilament tension. Clearly, functional studies in the intact myofilament are necessary to determine the contribution of these different TnI domains to PKA-induced changes in the myofilament Ca2+ sensitivity of tension.
Viral-mediated gene transfer to adult cardiac myocytes was used in the
present study to investigate the domain(s) within cTnI responsible for
changes in myofilament tension after PKA activation. This approach has
previously been shown to result in virtually complete replacement of
endogenous cTnI with the ssTnI isoform or individual TnI chimeras
without detectable changes in sarcomere architecture, contractile
protein stoichiometry, or the isoform expression pattern of other
contractile proteins (35, 36, 38). In addition, there is
no detectable influence on myofilament function after viral-based gene
transfer of the endogenous cTnI isoform (35, 36).
Collectively, these findings provide strong evidence for the
specificity of myofilament gene transfer. Importantly, gene transfer of
TnI isoform/chimeras has provided important new insights into the
function of TnI within intact myofilaments of adult myocytes (35,
36, 38). Measurements of myofilament tension in adult myocytes
expressing either ssTnI or one of the TnI chimeras constructed from
cTnI and ssTnI indicate that there is a hierarchy of myofilament
Ca2+ sensitivity of tension (35, 36, 38). This
hierarchy is best explained by the presence of separate amino and
carboxyl terminal TnI isoform-specific domains that influence
myofilament Ca2+ sensitivity of tension, whereas earlier
investigators predicted only a carboxyl terminal region influenced
Ca2+ sensitivity (10, 32, 34). A previously
unknown carboxyl terminal domain responsible for acidic pH-induced
decreases in submaximal tension also is observed (see Fig. 1). Acute
genetic engineering of TnI is now used here to analyze the domain(s)
within TnI mediating the PKA-induced decrease in myofilament
Ca2+ sensitivity of adult cardiac myocytes.
|
Experiments in the present study were designed to determine whether the isoform-specific amino terminal region of cTnI coupled to the carboxyl terminus of either TnI isoform is sufficient to account for the direction and magnitude of the PKA-mediated decrease in myofilament Ca2+ sensitivity. This possibility can now be differentiated from an alternative possibility that isoform-specific domains within the carboxyl terminal region influence these aspects of the myofilament tension response to PKA in adult myocytes. The relative contribution of an isoform-specific domain in the amino terminus of cTnI was evaluated by expressing a phosphorylatable TnI chimera (N-card/slow-C TnI), which contains the amino terminus of cTnI and carboxyl terminus of ssTnI (see Fig. 1), in adult myocytes. The shift in myofilament Ca2+ sensitivity caused by PKA in myocytes expressing this chimera was then compared with the shift observed in myocytes expressing cTnI, ssTnI, or a TnI chimera with the amino terminus of ssTnI, which lacks the amino terminal phosphorylation sites (N-slow/card-C TnI). Additional insight into the region(s) of TnI involved in the myofilament response to PKA was obtained by analyzing the desensitizing effects of PKA phosphorylation in the presence of acidic pH. The additive effect of PKA-mediated cTnI phosphorylation and acidosis observed in the present study signals that PKA operates via a region that is separate from the isoform-specific region of TnI affected by acidic pH.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mutagenesis Strategy
Full-length wild-type ssTnI and cTnI cDNAs were used to generate the TnI chimeras, as described in earlier studies (35, 36). The TnI chimera designated N-slow/card-C TnI (35) is composed of the amino terminus of ssTnI joined to the carboxyl portion of cTnI, and a second chimera (36), designated N-card/slow-C TnI, consists of the amino terminus of cTnI and the carboxyl terminus of ssTnI. The N-slow/card-C TnI chimera is composed of the 68 amino terminal amino acids of ssTnI and carboxyl terminal 110 amino acids of cTnI, whereas the 99 amino acids from the amino terminus of cTnI are joined to the carboxyl terminal 120 amino acids of ssTnI to form N-card/slow-C TnI. The alignment of the four proteins relative to one another is shown in Fig. 1.Generation of Adenoviral Vectors
Recombinant adenovirus vectors were constructed by cotransfecting shuttle plasmids containing TnI cDNAs (cTnI, ssTnI, N-slow/card-C TnI, and N-card/slow-C TnI) and pJM17 into HEK 293 cells as described in detail previously (37, 38). Recombinant virus was confirmed by Southern blot analysis (35, 36). High titers of plaque-purified adenoviral stocks were prepared from plaque-purified cellular lysates with a CsCl gradient followed by dialysis in PBS with 10% glycerol for 24 h. Aliquots of virus were stored atPrimary Cultures of Rat Ventricular Myocytes
Ventricular myocytes were isolated from adult female rats as described by Westfall et al. (37). An aliquot of Ca2+-tolerant myocytes (2 × 104 myocytes) was then plated on laminin-coated coverslips and incubated at 37°C in DMEM containing 5% FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin for 2 h. Cells were then incubated with recombinant adenovirus in DMEM plus penicillin/streptomycin. An aliquot of DMEM plus penicillin/streptomycin (2 ml) was added to each coverslip after 1 h, and serum-free medium was changed the day after adding virus and then every 2-3 days up to 7 days of culture.Phosphorylation of Myofilament Proteins in Permeabilized Cardiac Myocytes
Myocytes cultured for 6-7 days were permeabilized in 0.1% Triton X-100 for 1 min and rinsed three times in pCa 9.0 relaxing solution (RS; see below). Phosphorylation experiments were initiated by adding 75 µl of RS containing 10 µCi [Analysis of Protein Composition by Gel Electrophoresis and Western Blots
Gel electrophoresis. Cultured ventricular myocytes from coverslips were collected in sample buffer 6-7 days after plating and were separated by gel electrophoresis. Fiber segments of rat soleus and rabbit psoas muscles were collected as described previously (23). Samples labeled with 32P were boiled for 2 min, sonicated for 10 min, and briefly centrifuged. Proteins were then separated by SDS-PAGE and silver stained as previously described in detail (13). Dried gels were scanned (Scanmaker 4; Microtek), and individual radiolabeled protein bands were detected with a PhosphorImager. Scanned gels and the extent of phosphorylation were quantified using Multi-analyst software (Bio-Rad, Hercules, CA).
Western blot analysis. Cultured ventricular myocytes from coverslips were collected in sample buffer 6 days after plating and separated by gel electrophoresis as described above. Proteins were then transblotted onto a polyvinylidene difluoride membrane, and immunodetection was carried out on blots fixed in glutaraldehyde (39). TnI isoform/chimera composition was determined using a 1:4,000 dilution of the primary anti-TnI monoclonal antibody MAB 1691 (Chemicon, Temecula, CA), which recognizes all striated muscle isoforms from rat, as well as the two TnI chimeras (35, 36).
Measurement of Ca2+-Activated Tension in Single Cardiac Myocytes at pH 7.0 and 6.2
Solutions and preparation of samples for mechanical studies.
Complete details of the experimental chamber and attachment procedure
for mounting single, rod-shaped cardiac myocytes has been reported
elsewhere (23). The relaxing and activating solutions used
for experiments contained 7 mM EGTA, 20 mM imidazole, 1 mM free
Mg2+, 14.5 mM creatine phosphate, and 4 mM MgATP with
sufficient KCl to yield a total ionic strength of 180 mM. Solution pH
was adjusted to 7.00 or 6.20 with KOH/HCl. The RS had a pCa
(log[Ca2+]) of 9.0, whereas the pCa of the solution for
maximal activation was 4.0. The computer program of Fabiato
(9) was used to calculate the final concentrations of each
metal, ligand, and metal-ligand complex, employing the stability
constants listed by Godt and Lindley (14).
Measurement of steady-state isometric tension-pCa relationship. Ca2+-activated tension was measured in single myocytes by allowing steady-state isometric tension to develop at each pCa, followed by rapid slackening to obtain the tension baseline. Myocytes were subsequently returned to the original cell length and placed in RS. The difference between developed tension and the tension baseline after the slack step was measured as total tension. Active tension was obtained by subtracting resting tension measured at pCa 9.0 from total tension. Tension-pCa relationships were constructed by expressing tension at various submaximal Ca2+ concentrations as a fraction of tension at maximal activation (pCa 4.0). Every third or fourth activation was carried out at pCa 4.0.
Two general protocols were used in experiments with the catalytic subunit of PKA. In the first protocol (protocol 1), a coverslip containing the single myocytes was washed with RS, briefly exposed to RS with 0.1% Triton X-100 for 30-60 s, and then washed three times with standard RS. Phosphorylation was achieved by exposing myocytes to a RS with dithiothreitol (DTT, 6 mg/ml) plus 1 U/µl PKA (catalytic subunit) for ~25 min (range 15-60 min) at 25°C. Single myocytes were then attached to the recording apparatus as described above, and the tension-pCa relation was determined. In the second protocol (protocol 2), which was used more extensively in these studies, single myocytes were first attached to the recording apparatus, and the membrane was permeabilized as above. The tension-pCa relationship was determined at pH 7.0 and 6.2, the myocyte was exposed to RS containing PKA (see protocol 1) for 30 min, and then the tension-pCa relation at each pH was measured again. In this protocol, the myocyte serves as its own control (i.e., +/
![]() |
Statistics
Values for each group are expressed as means ± SE. ANOVA was used to test for significant differences between groups, with a post hoc Student-Newman-Keuls multiple comparison test to determine significance (P < 0.05). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Contribution of TnI to PKA-Mediated Decreases in Myofilament Ca2+ Sensitivity of Tension in Adult Rat Cardiac Myocytes
Investigation of the TnI domains involved in mediating PKA-induced decreases in myofilament Ca2+ sensitivity requires the retention of an intact PKA signaling pathway in primary cultures of adult myocytes, particularly in relation to PKA-mediated changes in Ca2+-activated tension. Previously, it has been shown that the thick filament protein, myosin-binding protein C (MyBP-C), is phosphorylated along with cTnI in response to PKA activation (18). Comparable results are observed in adult cardiac myocytes maintained in culture for 6 days, as seen by the phosphorylation of cTnI and MyBP-C by the catalytic subunit of PKA in membrane-permeabilized adult myocytes (Fig. 2) and by isoproterenol stimulation of intact myocytes (Fig. 3). In freshly isolated cardiac myocytes, PKA activation decreases myofilament Ca2+ sensitivity of tension (15), and this decrease in the Ca2+ sensitivity of myofilament tension is retained in myocytes maintained in culture for 6 days (Fig. 4A). Taken together, the functional and phosphorylation results provide strong evidence that the PKA signaling pathway is intact in cultured adult cardiac myocytes maintained in serum-free culture conditions.
|
|
|
The next step toward determining the TnI domain(s) involved in
PKA-mediated changes in myofilament function was to specifically replace endogenous cTnI with a nonphosphorylatable, exogenous TnI
within 6 days after TnI gene transfer. Western blot analysis demonstrated that ssTnI, which lacks the Ser-23/Ser-24 PKA
phosphorylation sites (1), is expressed in myocytes after
gene transfer with a corresponding decrease in endogenous cTnI
expression (Fig. 5), in agreement with
earlier findings (38). Previous studies also have directly
demonstrated that expressed exogenous ssTnI is incorporated in the
myofilaments of adult cardiac myocytes without detected changes in
contractile protein stoichiometry or in the architecture of the
sarcomere (38), thereby demonstrating the specificity of
this approach. Thus, observed changes in myofilament function are the
direct result of the newly expressed and incorporated ssTnI protein.
|
The pattern of myofilament protein phosphorylation produced in response
to the catalytic subunit of PKA was then studied 6 days after ssTnI
gene transfer in membrane-permeabilized myocytes. Both cTnI and MyBP-C
were phosphorylated in untreated and AdCMVcTnI-treated myocytes,
whereas only MyBP-C was phosphorylated in myocytes expressing the ssTnI
isoform (Fig. 2). Similar results were obtained in intact myocytes
treated with 100 nM isoproterenol (Fig. 3). Membrane-permeabilized myocytes expressing cTnI or ssTnI were then used to determine the
relative contribution of cTnI and MyBP-C phosphorylation (Fig. 2 and
Table 1) to PKA-mediated changes in
Ca2+-activated tension. Myofilament Ca2+
sensitivity of tension did not change significantly after PKA treatment
in myocytes expressing ssTnI, whereas PKA caused the anticipated
decrease in Ca2+ sensitivity in myocytes expressing cTnI
(Figs. 4 and 6). This PKA response is TnI
isoform specific and is not influenced by adenoviral gene transfer per
se, as demonstrated by the comparable PKA-mediated shifts in
pCa50 observed in "control" myocytes and AdCMVcTnI-treated myocytes (Fig. 6).
|
|
Separate experiments with the nonphosphorylatable TnI chimera, N-slow/card-C TnI, also were carried out with the expectation that the findings would be similar to those observed in ssTnI-expressing myocytes. As with ssTnI, this chimera was expressed in myocytes after gene transfer with a corresponding decrease in endogenous cTnI expression (Fig. 5). The pattern of TnI and MyBP-C phosphorylation in myocytes expressing the N-slow/card-C TnI chimera also matched the one obtained with myocytes expressing ssTnI (Fig. 2 and Table 1), and PKA treatment resulted in a shift in pCa50 comparable to myocytes expressing ssTnI (Figs. 4 and 6). Thus, although PKA phosphorylated cTnI and decreased myofilament Ca2+ sensitivity in myocytes, neither ssTnI nor N-slow/card TnI was phosphorylated, and, despite the continued phosphorylation of MyBP-C in myocytes expressing these exogenous TnI proteins, there was no significant change in myofilament Ca2+ sensitivity after PKA activation (Figs. 4 and 6). The tight relationship between PKA-induced TnI phosphorylation and decrease in myofilament Ca2+ sensitivity of tension indicates that TnI phosphorylation is the key event responsible for the PKA-mediated decrease in myofilament Ca2+ sensitivity.
Relative Contributions of TnI Domains to the PKA-Mediated Decrease in Myofilament Ca2+ Sensitivity of Tension
The role played by interactions between the amino and carboxyl portions of TnI during the PKA-mediated decrease in myofilament Ca2+ sensitivity was investigated in myocytes expressing the N-card/slow-C TnI chimera protein. This chimera contains the amino terminal extension of cTnI with the two primary PKA phosphorylation targets plus 67 additional amino terminal residues and the carboxyl region of ssTnI. Activation of PKA in permeabilized (Fig. 2) and intact (results not shown) adult cardiac myocytes resulted in phosphorylation of N-card/slow-C TnI. Total phosphate incorporation in this TnI chimera over 30 min was similar to that found with cTnI (Table 1). A rightward shift in myofilament Ca2+ sensitivity was observed after PKA activation in myocytes expressing the N-card/slow-C TnI, and the magnitude of this change was comparable to the shift observed with cTnI (Figs. 4 and 6). These results with N-card/slow-C TnI demonstrate that a region within the 99 amino terminal residues of cTnI (e.g., amino terminus) plays a significant role in the direction and magnitude of the PKA-induced shift in myofilament Ca2+ sensitivity. In addition, results obtained with the N-card/slow-C TnI chimera indicate that isoform-specific residues in the carboxyl terminal 120 amino acids of ssTnI (e.g., carboxyl terminus) have no significant effect on the magnitude of the tension response to PKA.Additive Effects of pH and Phosphorylation on Myofilament Ca2+ Sensitivity
The effects of PKA-mediated TnI phosphorylation and acidic pH on myofilament Ca2+ sensitivity of tension were examined as an alternative approach for defining the domain(s) responsible for the phosphorylated TnI-induced decreases in myofilament Ca2+ sensitivity. Acidic pH has been shown to decrease the Ca2+ sensitivity of myofilament tension (28), and recent studies with the N-card/slow-C and N-slow/card-C TnI chimeras provide strong evidence that the carboxyl terminal domain of TnI mediates this response (35, 36). Thus the condition of acidic pH can be introduced after PKA treatment to further determine whether the carboxyl terminus of cTnI influences the magnitude of the phosphorylation-induced decrease in myofilament Ca2+ sensitivity. In myocytes expressing cTnI, the rightward shift in the myofilament Ca2+ sensitivity of tension caused by acidic pH in the presence and absence of phosphorylation was similar in magnitude (Fig. 7A). A comparable shift in myofilament Ca2+ sensitivity also was observed when myocytes previously treated with PKA were exposed to pH 7.0 and acidic pH (Fig. 7B). Thus PKA-induced TnI phosphorylation and acidic pH additively decrease myofilament Ca2+ sensitivity and appear to act on TnI via independent mechanisms.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments with adult single cardiac myocytes expressing exogenous TnI proteins provide new evidence that the amino terminal region of TnI is an important domain responsible for the magnitude of the PKA-induced decrease in Ca2+ sensitivity of myofilament tension. Interestingly, the comparable functional effects of PKA observed with myocytes expressing cTnI or the N-card/slow-C TnI chimera (Fig. 6) demonstrate that isoform differences in the carboxyl terminus of TnI have no effect on the magnitude of the PKA-mediated decrease in myofilament Ca2+ sensitivity. Instead, these results point to an essential role of the amino portion of cTnI alone, or in combination with non-isoform-specific functional regions in the carboxyl terminus, in mediating PKA-induced alterations in cardiac mechanical function. Additionally, our study provides evidence that PKA-mediated cTnI phosphorylation works independently from acidic pH to shift myofilament Ca2+ sensitivity. Thus the present findings provide new insight into the specific functional domains within TnI in the intact myofilament of adult cardiac myocytes and further demonstrate the utility of this gene transfer approach for dissecting the functions of individual contractile proteins within adult myocytes.
The contribution of cTnI phosphorylation to PKA-mediated changes in
myofilament Ca2+ sensitivity appears to have important
clinical significance. Evidence is accumulating that basal and
PKA-mediated TnI phosphorylations change under several conditions known
to result in heart failure, including dilated cardiomyopathy (5,
24, 41), ischemic cardiomyopathy (2, 20), and
hypertension (22). There is also indirect evidence that
-adrenergic-mediated TnI phosphorylation plays an important role in
myocardial relaxation (29, 40). Thus the
cardiac response to changes in sympathetic tone may be mediated, in
part, by the ability of the TnI to be phosphorylated by PKA.
Role of TnI Domains in PKA-Mediated Decrease in Myofilament Ca2+ Sensitivity
The critical role of phosphorylated TnI in the PKA-mediated decrease in myofilament Ca2+ sensitivity, along with its potential clinical significance, makes it imperative to understand how TnI transduces the phosphorylation signal into a myofilament response. The phosphorylation event itself is relatively well worked out (reviewed in Ref. 30). Ser-23 and Ser-24 are the primary PKA targets (24, 33); phosphorylation of Ser-24 appears to precede Ser-23 phosphorylation in cTnI (25, 31), and phosphorylation of both serine residues is thought to be necessary for changes in myofilament function (31, 43). There are indications that PKA-induced TnI phosphorylation results in conformational changes within the amino terminal extension (17, 31). However, the molecular events that result from this modification of the amino terminal extension and that ultimately lead to a change in TnI regulation of contractile function remain controversial. Some investigators propose that functional changes caused by PKA phosphorylation are signaled directly through this amino terminal extension (12, 17), whereas others suggest that the Ca2+-sensitive region located in the carboxyl terminus of TnI also may be involved (6, 7). Isoform differences in the carboxyl terminus of TnI influence myofilament Ca2+ sensitivity of tension (refer to Fig. 1 and Refs. 35 and 36) and the magnitude of change in myofilament Ca2+ sensitivity of tension in response to variables such as acidosis (36). These isoform differences do not appear to be involved in determining the magnitude of the Ca2+ sensitivity decrease induced by PKA-mediated phosphorylation of TnI because the magnitude of the response is similar in myocytes expressing N-card/slow-C TnI and cTnI (Fig. 6). This conclusion does not rule out the possibility that the non-isoform-specific, Ca2+-sensitive regions within the carboxyl terminus of TnI may contribute to the direction, shape, and magnitude of the PKA-mediated change in the myofilament tension-pCa relationship. However, the known regions of TnI that bind other contractile proteins and influence Ca2+-sensitive actomyosin ATPase activity in solution studies, including the inhibitory peptide region and secondary actin and TnC binding domains [Tripet et al. (34)], each contain isoform-specific amino acid differences [Murphy et al. (27)]. Thus the amino terminus of TnI likely plays a significant role in mediating the conformational changes within TnI in response to PKA, although an as yet undefined non-isoform-specific, Ca2+-sensitive domain(s) in the carboxyl portion of TnI also may contribute to this response.The amino terminus includes the 32-amino acid extension specific for cTnI, which may function as a key domain in mediating the PKA-induced change in myofilament Ca2+ sensitivity in adult myocytes. This amino terminal extension would probably not function as an isolated peptide but would require the presence of a TnI backbone of cTnI or ssTnI origin. Alternatively, the PKA-mediated TnI phosphorylation response could be mediated via a second Ca2+-sensitive region previously described for the amino terminus of TnI (99 amino acids of N-card/slow-C TnI), which includes the 32-amino acid extension and binding domains for TnC and TnT (reviewed in Refs. 10 and 30). Future experiments with more specifically mutated regions within TnI will be needed to further define the critical interactions within the amino terminus that are necessary to transduce the TnI phosphorylation signal into the shift in myofilament Ca2+ sensitivity of tension.
Influences of pH and Phosphorylation on Myofilament Ca2+ Sensitivity
Myofilament tension measurements in myocytes treated with PKA and then exposed to acidic pH provide further evidence that TnI phosphorylation is likely mediated through the amino terminus of the protein. Acidic pH desensitizes myofilaments to Ca2+ via an isoform-specific, carboxyl terminal domain (35, 36). The combined effects of acidosis and PKA-mediated phosphorylation were previously studied in perfused rat hearts, with roughly additive effects observed on developed tension and relaxation (26). Acidosis was imposed during the phosphorylation period, and the additive effects of acidosis and phosphorylation were explained based on acidosis-induced increases in PKA-mediated TnI phosphorylation (26). In the present study, the catalytic subunit of PKA was removed before the introduction of acidic pH to avoid acidosis-induced changes in phosphorylation. The additive decrease in myofilament Ca2+ sensitivity observed with this protocol (e.g., acidosis after TnI phosphorylation; Fig. 7) is evidence that separate functional domains were responsible for acidosis and phosphorylation-mediated decreases in submaximal tension.Transduction Model for Phosphorylation-Induced Decreases in Myofilament Ca2+ Sensitivity
Overall, our results provide new information toward an understanding of the molecular mechanism whereby PKA-mediated TnI phosphorylation leads to a decrease in myofilament Ca2+ sensitivity. Previous investigators have obtained evidence that the phosphorylation of Ser-23/Ser-24 causes the 32-residue amino terminal extension of cTnI to act as a spacer arm that folds upon phosphorylation (1, 7) and increases the separation distance between TnI and TnC (21) such that there is altered Ca2+ binding to regulatory sites on TnC (16). Our results support a mechanism where the amino terminal extension of TnI appears to transduce this phosphorylation signal by interacting directly with TnC, as previously suggested by Al-Hillawi and colleagues (1). Results that support this view include PKA-induced shifts in myofilament Ca2+ sensitivity that are similar in magnitude in myocytes expressing TnI proteins with different isoform-specific carboxyl terminal regions and the additive effects of acidic pH and TnI phosphorylation on myofilament Ca2+ sensitivity of tension. An alternative proposal is that folding of the amino terminus in response to PKA induces a global conformational change in cTnI that is mainly transduced through the carboxyl terminus (6, 7). This mechanism would require the presence of an isoform-independent, Ca2+-sensitive region within the carboxyl terminus of TnI based on the similar PKA-induced decreases in Ca2+ sensitivity of tension in myocytes expressing cTnI and N-card/slow-C TnI. In the future, it will be important to determine whether phosphorylation of this amino terminal extension acts primarily as a spacer arm to physically decrease TnI-TnC interactions or induces conformational changes within TnC that are then transmitted to the regulatory Ca2+-binding sites in the amino terminus of TnC.In summary, experiments presented here establish the importance of the amino terminal region of cTnI in mediating the direction and magnitude of decreased myofilament Ca2+ sensitivity after PKA activation. With the use of gene transfer approaches, hypotheses addressing the relative function of the 32-amino acid extension of cTnI vs. the additional 67 amino terminal residues present in N-card/slow-C TnI during PKA activation can now be designed to gain more knowledge about the function of TnI during this signaling process within the context of the intact myofilament.
![]() |
ACKNOWLEDGEMENTS |
---|
We appreciate helpful comments from Dan Michele and Philip Wahr on earlier versions of the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by grants from the National Institutes of Health and American Heart Association to J. M. Metzger. J. M. Metzger is an Established Investigator of the American Heart Association, and M. V. Westfall was the recipient of a Scientist Development grant from the American Heart Association.
Address for reprint requests and other correspondence: M V Westfall, Dept of Physiology, Univ of Michigan, 1301 E Catherine St, 7712 Medical Sciences II, Ann Arbor, MI 48109-0622 (E-mail: wfall{at}w.imap.itd.umich.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 8 June 2000; accepted in final form 8 September 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Al-Hillawi, E,
Bhandari DG,
Trayer HR,
and
Trayer IP.
The effects of phosphorylation of cardiac troponin-I on its interactions with actin and cardiac troponin-C.
Eur J Biochem
228:
962-970,
1995[Abstract].
2.
Bartel, S,
Karczewski P,
and
Krause E-G.
Phosphorylation of phospholamban and troponin I in the ischemic and reperfused heart: attenuation and restoration of isoprenaline responsiveness.
Biomed Biochim Acta
48:
S108-S113,
1989[ISI][Medline].
3.
Bartel, S,
Morano I,
Hunger HD,
Katus H,
Pask HT,
Karczewski P,
and
Krause E-G.
Cardiac troponin I and tension generation of skinned fibres in the developing rat heart.
J Mol Cell Cardiol
26:
1123-1131,
1994[ISI][Medline].
4.
Bartel, S,
Stein B,
Eschenhagen T,
Mende U,
Neumann J,
Schmitz W,
Krause E-G,
Karczewski P,
and
Scholz H.
Protein phosphorylation in isolated trabeculae from nonfailing and failing human hearts.
Mol Cell Biochem
157:
171-179,
1996[ISI][Medline].
5.
Bodor, GS,
Oakeley AE,
Allen PD,
Crimmins DL,
Ladenson JH,
and
Anderson PAW
Troponin I phosphorylation in the normal and failing adult human heart.
Circulation
96:
1495-1500,
1997
6.
Chandra, M,
Dong WJ,
Pan BS,
Cheung HC,
and
Solaro RJ.
Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C.
Biochemistry
36:
13305-13311,
1997[ISI][Medline].
7.
Dong, W-J,
Chandra M,
Xing J,
She M,
Solaro RJ,
and
Cheung HC.
Phosphorylation-induced distance change in a cardiac muscle troponin I mutant.
Biochemistry
36:
6754-6761,
1997[ISI][Medline].
8.
England, PJ.
Correlation between contraction and phosphorylation of the inhibitory subunit of troponin in perfused rat heart.
FEBS Lett
50:
57-60,
1975[ISI][Medline].
9.
Fabiato, A.
Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands.
Methods Enzymol
157:
378-417,
1988[ISI][Medline].
10.
Farah, CS,
and
Reinach RC.
The troponin complex and regulation of muscle contraction.
FASEB J
9:
755-767,
1995
11.
Fentzke, RC,
Buck SH,
Patel JR,
Lin H,
Wolska BM,
Stojanovic MO,
Martin AF,
Solaro RJ,
Moss RL,
and
Leiden JM.
Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart.
J Physiol (Lond)
517:
143-157,
1999
12.
Gaponenko, V,
Abusamhadneh E,
Abbott MB,
Finley N,
Gasmi-Seabrook G,
Solaro RJ,
Rance M,
and
Rosevear PR.
Effects of troponin I phosphorylation on conformational exchange in the regulatory domain of cardiac troponin C.
J Biol Chem
274:
16681-16684,
1999
13.
Giulian, GG,
Moss RL,
and
Greaser ML.
Improved methodology for the analysis and quantitation of proteins on one-dimensional silver-stained slab gels.
Anal Biochem
129:
277-287,
1983[ISI][Medline].
14.
Godt, RE,
and
Lindley BD.
Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog.
J Gen Physiol
80:
279-297,
1982[Abstract].
15.
Hofmann, PA,
and
Lange JH.
Effects of phosphorylation of troponin I and C protein in isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats.
Circ Res
74:
716-726,
1994.
16.
Holroyde, MJ,
Howe E,
and
Solaro RJ.
Modification of calcium requirements for activation of cardiac myofibrillar ATPase by cyclic AMP dependent phosphorylation.
Biochim Biophys Acta
586:
63-69,
1979[ISI].
17.
Jaquet, K,
Lohmann K,
Czisch M,
Holak T,
Gulati J,
and
Jaquet R.
A model for the function of the bisphosphorylated heart-specific troponin I N-terminus.
J Muscle Res Cell Motil
19:
647-659,
1998[ISI][Medline].
18.
Jeacocke, S,
and
England P.
Phosphorylation of a myofibrillar protein of Mr 150,000 in perfused rat heart, and the tentative identification of this as C-protein.
FEBS Lett
122:
129-132,
1980[ISI][Medline].
19.
Koppe, RI,
Hallauer PL,
Karpati G,
and
Hastings KEM
cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs.
J Biol Chem
264:
14327-14333,
1989
20.
Li, P,
Hofmann PA,
Li B,
Malhotra A,
Cheng A,
Sonnenblick EH,
Meggs LG,
and
Anversa P.
Myocardial infarction alters myofilament calcium sensitivity and mechanical behavior of myocytes.
Am J Physiol Heart Circ Physiol
272:
H360-H370,
1997
21.
Liao, R,
Wang C-K,
and
Cheung HC.
Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle.
Biochemistry
33:
12729-12734,
1994[ISI][Medline].
22.
McConnell, BK,
Moravec CS,
and
Bond M.
Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy.
Am J Physiol Heart Circ Physiol
274:
H385-H396,
1998
23.
Metzger, JM.
Myosin binding-induced cooperative activation of the thin filament in cardiac myocytes and skeletal muscle fibers.
Biophys J
68:
1430-1442,
1995[Abstract].
24.
Mittmann, K,
Jaquet K,
and
Heilmeyer LMG, Jr.
A common motif of two adjacent phosphoserines in bovine, rabbit and human cardiac troponin I.
FEBS Lett
273:
41-45,
1990[ISI][Medline].
25.
Mittman, K,
Jaquet K,
and
Heilmeyer LMG, Jr.
Ordered phosphorylation of a minimal recognition motif for cAMP dependent protein kinase present in cardiac troponin I.
FEBS Lett
302:
133-137,
1992[ISI][Medline].
26.
Mundina-Weilenmann, C,
Vittone L,
Cingolani HE,
and
Orchard CH.
Effects of acidosis on phosphorylation of phospholamban and troponin I in rat cardiac muscle.
Am J Physiol Cell Physiol
270:
C107-C114,
1996
27.
Murphy, A,
Jones L, II,
Sims HF,
and
Strauss AW.
Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart.
Biochemistry
30:
707-712,
1991[ISI][Medline].
28.
Orchard, CH,
and
Kentish JC.
Effects of changes of pH on the contractile function of cardiac muscle.
Am J Physiol Cell Physiol
258:
C967-C981,
1990
29.
Pan, B-S,
Hannon JD,
Wiedmann R,
Potter JD,
Kranias EG,
Shen Y-T,
Johnson RG, Jr,
and
Housmans PR.
Effects of isoproterenol on twitch contraction of wild type and phospholamban-deficient murine ventricular myocardium.
J Mol Cell Cardiol
31:
159-166,
1999[ISI][Medline].
30.
Perry, SV.
Troponin I: inhibitory or facilitator.
Mol Cell Biochem
190:
9-32,
1999[ISI][Medline].
31.
Quirk, PG,
Patchell VB,
Gao Y,
Levine BA,
and
Perry SV.
Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin I: structure-activity implications of ordered phosphorylation.
FEBS Lett
370:
175-178,
1995[ISI][Medline].
32.
Rarick, HM,
Tu X,
Solaro RJ,
and
Martin AF.
The C terminus of cardiac troponin I is essential for full inhibitory activity and Ca2+ sensitivity of rat myofibrils.
J Biol Chem
272:
26887-26892,
1997
33.
Swiderek, K,
Jaquet K,
Meyer HE,
Schachtele C,
Hofmann F,
and
Heilmeyer LMG, Jr.
Sites phosphorylated in bovine cardiac troponin T and I.
Eur J Biochem
190:
575-582,
1990[Abstract].
34.
Tripet, B,
Van Eyk JE,
and
Hodges RS.
Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction.
J Mol Biol
271:
728-750,
1997[ISI][Medline].
35.
Westfall, MV,
Albayya FP,
and
Metzger JM.
Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes.
J Biol Chem
274:
22508-22516,
1999
36.
Westfall, MV,
Albayya FP,
Turner II,
and
Metzger JM.
Chimera analysis of troponin I domains that influence Ca2+-activated myofilament tension in adult cardiac myocytes.
Circ Res
86:
470-477,
2000
37.
Westfall, MV,
Rust EM,
Albayya F,
and
Metzger JM.
Adenovirus-mediated myofilament gene transfer into adult cardiac myocytes.
Methods Cell Biol
52:
307-322,
1998[ISI].
38.
Westfall, MV,
Rust EM,
and
Metzger JM.
Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function.
Proc Natl Acad Sci USA
94:
5444-5449,
1997
39.
Westfall, MV,
Samuelson LC,
and
Metzger JM.
Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.
Dev Dyn
206:
24-38,
1996[ISI][Medline].
40.
Wolska, BM,
Stojanovic MO,
Luo W,
Kranias EG,
and
Solaro RJ.
Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+.
Am J Physiol Cell Physiol
271:
C391-C397,
1996
41.
Zakhary, DR,
Moravec CS,
Stewart RW,
and
Bond M.
Protein kinase A (PKA)-dependent troponin I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy.
Circ Res
99:
505-510,
1999.
42.
Zhang, R,
Zhao J,
Mandveno A,
and
Potter JD.
Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation.
Circ Res
76:
1028-1035,
1995
43.
Zhang, R,
Zhao J,
and
Potter JD.
Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C.
J Biol Chem
270:
30773-30780,
1995