1 Department of Physiology and Biophysics and 2 Section of Cardiology, Department of Medicine, Program in Cardiovascular Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation
of protein kinase C (PKC) in heart muscle signals hypertrophy and may
also directly affect contractile function. We tested this idea using a
transgenic (TG) mouse model in which conditionally expressed PKC was
turned on at 10 wk of age and remained on for either 6 or 10 mo.
Compared with controls, TG cardiac myocytes demonstrated an increase in
the peak amplitude of the Ca2+ transient, an increase in
the extent and rate of shortening, and an increase in the rate of
relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation
and Ca2+-uptake rates of sarcoplasmic reticulum vesicles
were the same in TG and control heart preparations. At 10 mo, TG
skinned fiber bundles demonstrated the same sensitivity to
Ca2+ as controls, but maximum tension was depressed and
there was increased myofilament protein phosphorylation. Our results
differ from studies in which PKC
was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca2+ transient.
hypertrophy; signal transduction; myofilaments; conditional transgenic; protein kinase C
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN
EXPERIMENTS PRESENTED HERE, we used a binary transgenic (TG)
mouse model in which a constitutively active -isoform of protein
kinase C (PKC) was conditionally expressed at a low level in the heart.
PKC
is normally expressed in fetal heart, declines after birth
(20, 21), and remains low in abundance throughout normal
adult life (2). There are, however, several lines of evidence indicating that the PKC
isoform is upregulated during development of cardiac hypertrophy. Hypertrophy induced by pressure overload in rats is associated with an increased level of PKC
isoform expression (12). Moreover, samples of failed human
heart demonstrate an increase in PKC
expression and an increase in its contribution to total PKC activity (3).
To test the hypothesis that upregulation is an important element in the
hypertrophic pathway, increased levels of the PKC isoform have been
induced using TG approaches. Wakasaki et al. (27) reported
that cardiac- specific and robust overexpression of the PKC
isoform
in TG mice induced a cardiomyopathy, which was characterized by left
ventricular hypertrophy, myocardial fibrosis, and decreased in vivo
left ventricular performance. However, Bowman et al. (4),
who employed conditional and relatively low expression of the PKC
transgene in adult mice, reported that modest upregulation caused mild
and progressive ventricular hypertrophy without significant
pathological changes. Compared with controls, these hearts did,
however, exhibit reduced rates of rise and fall of left ventricular
pressure in open-chest preparations. In contrast, overexpression of
PKC
in newborns resulted in sudden death marked by abnormalities in
the regulation of intracellular Ca2+ in myocytes. In the
case of mouse hearts subjected to high levels of constitutive
expression of the PKC
isoform, Takeishi et al. (26)
reported that myocyte shortening was depressed with no change in the
peak amplitude of the Ca2+ transients. Associated with this
effect was an increase in troponin I (TnI) phosphorylation. This
finding led to the speculation that a reduction in myofilament
activation, previously shown to be caused by PKC-dependent
phosphorylation of cardiac troponin I (cTnI) (14, 18, 19),
may be a critical mechanism for heart failure. Thus the mechanism by
which PKC
may be involved in the hypertrophic-failure process
appears to depend on the timing of the upregulation of the enzyme as
well as on variable effects of upregulation on Ca2+
homeostasis and on myofilament response to Ca2+.
To test this idea, we induced expression of PKC in TG mice at 10 wk
of age and studied ventricular preparations at 6 and 10 mo of age. We
compared preparations from controls and TG hearts with regard to
1) mechanical function and intracellular Ca2+
transients of ventricular myocytes, 2) protein
phosphorylation profiles of cellular proteins, 3)
Ca2+ dependence of tension in skinned fiber bundles, and
4) Ca2+ transport rate of sarcoplasmic reticulum
(SR) vesicles. Our results indicate that low-level expression of
PKC
, which may mimic a potential early response to upregulation in
hypertrophy, induced changes in cellular Ca2+ regulation
that are quite different from those seen in later, more malignant,
stages of the pathology.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TG animals.
Conditional expression of a constitutively active PKC isoform was
carried out using a tetracycline-controlled promoter (4, 11). PKC
expression was turned on at the age of 10 wk in
binary TG [tTA/PKC
, which we will refer to as TG (+/+)]. Controls
were wild-type mice or single TG, tTA/PKC
(+/
), that do not
express PKC
. As previously reported (4), preparations
from single (+/
) and nontransgenic (NTG) mouse hearts differ in no
significant way. The genotype of each animal was determined by either
PCR or Southern blotting. Results of comparative determinations of the
overall PKC activity indicate that the TG hearts have an ~10% increase in activity over the controls. Quantitative RT-PCR (using primers with matched transmembrane segments and real-time fluorescent PCR) has shown that the message level of the transgene was
approximately five- to tenfold higher than the endogenous gene and that
expression of the transgene does not change the level of expression of
the wild-type genes.
Isolation of myocytes. Adult mice were heparinized (5,000 U/kg body wt), and, after 30 min, anesthetized with ether. Left ventricular myocytes were isolated as previously described (32) and were studied 1-6 h after isolation. The cells used for phosphorylation experiments were sequentially resuspended in 0.2, 0.5, and 1.0 mmol/l Ca2+ in a sodium HEPES phosphate-free buffer (pH 7.4) of the following composition (in mmol/l): 4.8 KCl, 1.2 MgSO4, 132 NaCl, 10 HEPES, 2.5 sodium pyruvate, and 10 glucose.
Measurement of intracellular Ca2+ transients and cell shortening. After isolation, the cells were loaded for 15 min at room temperature with 3 µmol/l fura 2-AM in Tyrode solution (0.5 mmol/l Ca2+) with 1 mg/ml bovine serum albumin and 5% fetal bovine serum. Fura 2 fluorescence and cell shortening were monitored simultaneously, as described in detail by Wolska et. al (33). Most cells were stimulated at 0.5 Hz, but in one series of experiments, we measured cell shortening over a frequency range of 0.25-3 Hz, with stimulation at each frequency for 30 s, at which time the contractions were stable.
Labeling of mouse myocytes with 32P and determination
of protein phosphorylation.
The level of protein phosphorylation in myocyte preparations was
measured using a protocol modified after that described by Wolska et
al. (33). Myocytes were incubated for 1 h in a
phosphate-free sodium HEPES buffer that contained 1 mM
CaCl2 and 0.250 mCi [32P]orthophosphate.
Myocytes were washed twice and solubilized in 40 µl of 1% SDS
containing 3 mM EDTA and sonicated for 2 min. The samples were boiled
for 10 min before 12.5% SDS-PAGE. Each lane was loaded with 25 µg of
protein. The gels were stained for 2 h. Destained gels were
exposed to a phosphor screen overnight using STORM (Molecular Dynamics)
and ImageQuant software to determine the 32P incorporation
after background correction. We used a densitometric scan of the
Coomassie blue-stained gel to test for equal loading of the
myofilament proteins. Actin and tropomyosin were used as standards and
were not statistically different in the NTG and both TG lanes.
Therefore, we concluded that any changes in the phosphorylation of the
myofilament proteins were the result of increased phosphorylation by
PKC.
Force measurements on skinned fiber bundles and
Ca2+ uptake by SR vesicles.
Measurements of the relationship between pCa (log of the molar free
Ca2+) and tension (force/cross-sectional area) were
performed on detergent-extracted fiber bundles as described in detail
previously (8, 31). The only modification was addition of
the phosphatase inhibitor calyculin A (0.1 µM) to the buffers.
Ca2+ uptake into SR vesicles in cardiac homogenates was
measured with the aid of 45CaCl2 using a
Millipore filtration assay as previously described (8,
22).
Statistical analysis. Data are presented as means ± SE. The Student's t-test was used for unpaired observations and one-way ANOVA for multiple comparison. P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell shortening and Ca2+ transients
in myocytes.
Morphological and mechanical properties, as well as stability of the
Ca2+ transients, were used as criteria for the viability
and stability of the myocytes. Before making measurements, all myocytes
were stimulated at 0.5 Hz for at least 20 min or until the shortening was stable for 15-20 min. Representative recordings of myocyte mechanics and Ca2+ transients in cells from
6-mo-old control and TG mice are shown in Fig.
1. As summarized in Table
1, the percentage of the extent of
myocyte shortening (%l), as well as the rate of shortening and rate of
relengthening, was significantly (P < 0.01) increased in TG (+/+)
myocytes compared with controls. Table 1 also indicates that the time
required for the cells to achieve 80% of full relaxation from the peak
of the shortening signal was reduced significantly in the TG (+/+) mice
compared with controls. Table 2 reports data indicating that the amplitude of the Ca2+ transient
was significantly increased in the TG (+/+) myocytes. The baseline
Ca2+ level was not different between control and TG (+/+)
mice. There was also a significant reduction in the time to 50% decay
of the Ca2+ signal from its peak in the TG (+/+) mice
compared with controls (Table 2). These data show that myocytes
isolated from hearts conditionally expressing low level PKC
demonstrated an increase in contractility.
|
|
|
|
|
Myofilament tension generation.
Figure 3 shows the pCa-tension relations
of skinned fiber bundles prepared from 10-mo-old binary TG (+/+) and
control mouse ventricles at sarcomere length 2.3 µm. Tension
generated by these two groups, which was measured in the presence of
the phosphatase inhibitor calyculin A, was equally sensitive to
Ca2+. The pCa50 for binary TG (+/+)
preparations was 5.54 ± 0.01 (n = 13 from 4 different hearts) and 5.53 ± 0.02 (n = 13 from 4 different hearts) for controls. However, maximum tension was
significantly reduced (P < 0.01) in the preparations
from binary mice (34.6 ± 1.6 mN/mm2) compared with
the controls (46.6 ± 2.2 mN/mm2). If both
preparations were permitted to dephosphorylate, this difference in
maximum tension was no longer evident (data not shown). Thus we cannot
ascribe the difference in maximum tension to a difference in the myosin
content of the preparations.
|
Phosphorylation of cardiac proteins.
To determine whether differences in covalent modulation among groups,
NTG (/
), TG binary (+/+), and single TG (+/
) could account for
the decrease in maximum tension, we measured phosphorylation of the
cellular proteins. Figure 4 shows an
autoradiogram of the cell proteins from the three groups. There were
significant increases in the 32P incorporation into myosin
binding protein C (MyBPC; 25%), troponin T (TnT; 39%), TnI (20%),
and myosin light chain 2 (MLC2; 60%) in the binary (+/+) TG myocyte
preparations, compared with the wild-type (
/
) and the single TG
(+/
), which were not different from each other. On the other hand,
there was no difference in the incorporation of 32P into
phospholamban.
|
SR Ca2+-uptake rates.
The effects of PKC expression on the initial rates of ATP-dependent SR
Ca2+ uptake facilitated with oxalate were assessed using
cardiac homogenates. Figure 5 shows the
initial rates of Ca2+ uptake by vesicles of SR, which were
assayed at various Ca2+ concentrations. The maximum uptake
rate by the SR vesicles of TG mouse hearts (421.9 ± 28.1 nmol · mg1 · min
1) was not
significantly different than the uptake rate of controls (441.5 ± 27.4 nmol · mg
1 · min
1).
EC50 values for Ca2+ dependence of uptake rates
were 6.49 ± 0.06 µM for controls and 6.51 ± 0.05 µM for
TG.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using a TG model that allows conditional expression of PKC in the
heart, we have been able to shed light on the genesis of hypertrophic
process and postulate how failure may follow. The major novel finding
of our study is that physiologically relevant, cardiac-specific
expression of PKC, initiated in adult life, produces significant
inotropic enhancement, as measured by unloaded rates of cell shortening
and relaxation. These changes are mirrored by increases in the dynamics
and peak amplitude of the Ca2+ transient and are associated
with significant cellular hypertrophy. It is also apparent that these
changes occur against a background of reduced maximum
tension-generating capability of the myofilaments with no change in
myofilament Ca2+ sensitivity. This scenario is in sharp
contrast with the changes induced by constitutive overexpression of
PKC in which there is a 20-fold increase in expression of
cytoplasmic PKC and a 10-fold increase in bound PKC (26,
27). These models demonstrate high levels of PKC expression and
activity from birth through adult life and induce depressed myocyte
contractility without change in the dynamics of the Ca2+
transient. Together, these two lines of evidence indicate that there
may be a hierarchy of PKC effects in the adult cardiocyte, initially
characterized by a balance of increased cellular Ca2+
availability and depressed myofilament activity in association with
mild, well-compensated hypertrophy. Our data also indicate that the
balance of responses to PKC activation may change in favor of depressed
myofilament activity with the progression from compensated hypertrophy
to decompensated heart failure.
The cellular address where enhancement of Ca2+ fluxes
resides appears to be the sarcolemma and not the SR, inasmuch as we
were unable to demonstrate altered rates of Ca2+ uptake by
vesicles of the SR in preparations from TG hearts. We cannot, however,
rule out a possible effect of PKC on Ca2+ release via the
ryanodine receptor (13, 16). L-type Ca2+
channels are likely candidates for the action of PKC. There are
several reports demonstrating that PKC phosphorylation enhances Ca2+ influx during the action potential. Moreover, PKC
activators, such as phorbol esters and diacylglycerols, increase
Ca2+-channel currents in cardiac and smooth muscle cells of
various mammals (6, 34). In addition, our own data from
studies on neonatal cardiocytes expressing the PKC
transgene
demonstrate both a prolongation of the Ca2+ transient
(4) and also an increase in the Ca2+
conductance through the L-type Ca2+ channel that is blocked
by a specific PKC
2 antagonist (1).
An alternate mechanism by which PKC might affect contractility is by PKC-dependent phosphorylation of the myofilament proteins cTnI, cardiac TnT (cTnT), MLC2, and MyBPC (23). Our results represent the first explicit determination of the net effect of protein phosphorylation on myofilament response to Ca2+ in a TG model of PKC expression. Our data show a depression in maximum tension with no effect on Ca2+ sensitivity. Earlier studies, including our own, have investigated effects of specific phosphorylation of the main sites: cTnI, cTnT, MyBPC, and MLC2. Among these sites, PKC-dependent phosphorylation of only two, cTnI and cTnT, have been reported to depress maximum actin-activated myosin ATPase rate and maximum cross-bridge binding to the thin filament, even at saturating Ca2+ (14, 18, 19). Also, phosphorylation of MLC2 has no effect on maximum tension (17). Phosphorylation of TnI may reduce Ca2+ sensitivity (29), and phosphorylation of MLC2 may increase Ca2+ sensitivity of force (17) and thus have offsetting effects. This finding is significant in that it demonstrates that when both are phosphorylated, covalent modulation of cTnI may outweigh the impact of modulation of MLC2 and impose a depression of myofilament of Ca2+ sensitivity. We (9, 29) could find no effect of MyBPC-protein phosphorylation on Ca2+ sensitivity or ATPase and maximum tension. On the other hand, phosphorylation of MyBPC may possibly be related, in part, to the enhanced rate of shortening of the isolated myocytes (30).
Our data indicate that at different stages of cardiac hypertrophy,
there is a shifting balance between the positive effects of PKC on the
Ca2+-channel conductance and the depressive effects of PKC
activation on the myofilaments. Our conclusion from the studies
presented here is that the increase in the amplitude of the
Ca2+ transient overrides the depression in maximum tension.
The depression in maximum tension was an ~25% decrease, whereas
there was an 85% increase in the rate of cell shortening
(+dL/dt) of the myocytes and an increase of 160%
in the amplitude of the Ca2+ transient. The steepness of
the relationship between Ca2+ and tension would indicate
that this increase in Ca2+ delivery to the myofilaments
would indeed increase contractility, despite a blunted maximum
tension. It remains a distinct possibility that the increased
Ca2+ could also trigger and promote the other features of
the hypertrophic phenotype, including PKC activation of phosphorylation
cascades affecting transcription (25). In models of heart
failure in which sustained activation of PKC can be demonstrated
(including prolonged insulin-deficient diabetes, end-stage
cardiomyopathy, and TG constitutive expression of PKC), we propose that
this balance may be tilted in favor of the negative inotropic effects
of myofilament protein phosphorylation, especially of cTnI and cTnT.
This proposal fits with data of de Tombe (7), who reported
that the early hypertrophic response following myocardial infarction in
rats was associated with a normal Ca2+-tension relationship
in skinned trabeculae. However, aftersigns of congestive heart
failure had become manifest; the Ca2+-tension
relationship was rightshifted and depressed, compared with
sham-operated controls. Moreover, previous studies (4) comparing controls with the TG model employed in the present study reported a depression in maximum pressure development in left ventricles of open-chest mice 9 mo after the PKC gene was turned on.
This result fits with our finding (Fig. 3) of a depression in maximum
tension-generating capability of the myofilaments 10 mo after PKC
was turned on.
What is not directly addressed in the present study is the role played
by specific isoforms of PKC. In the adult mouse heart, the prominent
isoforms are Ca2+ independent ( and
), although
Ca2+-dependent isoforms are also seen (24).
Whether the
-isoform is expressed in nonpathological adult cardiac
myocytes is controversial. However, during fetal life and in response
to pathological loads, increased expression is seen (10).
The cellular functions subserved by individual isoforms are not
established. PKC
appears to translocate to the sarcomere when
activated and has been shown to have a high affinity for TnI
(14). In contrast, phosphorylation of the L-type Ca2+ channel has been linked to the
Ca2+-dependent
- and
-isoforms (35).
Despite extensive data from in vivo and in vitro experiments linking
PKC activation to the development of pathological cardiac adaptations,
the mechanism(s) by which kinase activation induces a hypertrophic
response remains elusive. PKC influences a number of intracellular
processes, including transcriptional transactivation of late response
genes (25), alterations in cellular Ca2+
fluxes (5, 15, 28), and altered response of the
myofilaments to Ca2+ (11, 14, 19). Because it
is difficult to recapitulate the in vivo situation on isolated cells or
protein preparations, defining the relevance of each of these effects
has been aided greatly by TG approaches. We have investigated a novel
TG mouse model to establish that low levels of PKC activation in the
adult cardiocyte are sufficient to cause cardiac hypertrophy. The
hypertrophy is characterized by an increase in the amplitude of the
Ca2+ transient and increased rates of cell shortening,
despite a depression of maximum tension-generating capability by the
myofilaments. In contrast, more substantial increases in transgene
expression and enzyme activation have been reported to induce reduced
rates of cell shortening and a rightshifted Ca2+-tension
relationship associated with in vivo evidence of heart failure
(26). Our findings support the hypothesis that there is a
hierarchy of effects of enzyme activation in the cardiocyte and that
the progression from compensated to uncompensated hypertrophy and
failure may be a reflection of the intensity or duration of activation
of this second messenger pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Ron McKinney for technical assistance in carrying out these experiments.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Research Grants R37-HL-22231 (to R. J. Solaro), R01-HL-64035 (to R. J. Solaro), R01-HL-52230 (to P. M. Buttrick), and R29-HL-58591 (to B. M. Wolska). L. Huang and E. M. Burkart were supported in part by National Institutes of Health Training Grant T32-07692. E. M. Burkart is also the recipient of an American Heart Association Midwest Affiliate predoctoral fellowship.
Address for reprint requests and other correspondence: R. J. Solaro, Dept. of Physiology and Biophysics (M/C 901), College of Medicine, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342 (E-mail: solarorj{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 October 2000; accepted in final form 15 December 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alden, KJ,
Goldspink PH,
Buttrick PM,
and
Garcia J.
Increased Ca2+ flux by protein kinase C-2 and cardiac hypertrophy (Abstract).
Circulation
102:
II-192,
2000.
2.
Bogoyevitch, MA,
Parker PJ,
and
Sugden PH.
Characterization of protein kinase C isotype expression in adult rat heart.
Circ Res
72:
757-767,
1993[Abstract].
3.
Bowling, N,
Walsh RA,
Song G,
Estridge T,
Sandusky GE,
Fouts RL,
Mintze K,
Pickard T,
Roden R,
Bristow MR,
Sabbah HN,
Mizrahi JL,
Gromo G,
King GL,
and
Vlahos CJ.
Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart.
Circulation
99:
384-391,
1999
4.
Bowman, JC,
Steinberg SF,
Jiang T,
Geenen DL,
Fishman GI,
and
Buttrick PM.
Expression of protein kinase C in the heart causes hypertrophy in adult mice and sudden death in neonates.
J Clin Invest
100:
2189-2195,
1997
5.
Capogrossi, MC,
Kaku T,
Filburn CR,
Pelto DJ,
Hansford RG,
Spurgeon HA,
and
Lakatta EG.
Phorbol ester and dioctanoylglycerol stimulate membrane association of protein kinase C and have a negative inotropic effect mediated by changes in cytosolic Ca2+ in adult rat cardiac myocytes.
Circ Res
66:
1143-1155,
1990[Abstract].
6.
Chik, CL,
Li B,
Ogiwara T,
Ho AK,
and
Karpinski E.
PACAP modulates L-type Ca2+ channel currents in vascular smooth muscle cells: involvement of PKC and PKA.
FASEB J
10:
1310-1317,
1996
7.
De Tombe, PP.
Altered contractile function in heart failure.
Cardiovasc Res
37:
367-380,
1998[ISI][Medline].
8.
Evans, C,
Pena JR,
Muthuchamy M,
Wieczorek DF,
Solaro RJ,
and
Wolska BM.
Altered hemodynamics and response to -adrenergic stimulation in transgenic mice harboring a mutant tropomyosin linked to hypertrophic cardiomyopathy.
Am J Physiol Heart Circ Physiol
279:
H2414-H2423,
2000
9.
Fentzke, RC,
Buck SH,
Patel JR,
Lin H,
Wolska BM,
Stojanovic MO,
Martin AM,
Solaro RJ,
Moss RL,
and
Leiden JM.
Impaired cardiomycyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart.
J Physiol (Lond)
517:
143-157,
1999
10.
Goldberg, M,
and
Steinberg SF.
Tissue-specific developmental regulation of protein kinase C isoforms.
Biochem Pharmacol
51:
1089-1093,
1996[ISI][Medline].
11.
Gossen, M,
and
Bujard H.
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.
Proc Natl Acad Sci USA
89:
5547-5551,
1992[Abstract].
12.
Gu, X,
and
Bishop SP.
Increased protein kinase C and isozyme redistribution in pressure-overloaded cardiac hypertrophy in the rat.
Circ Res
75:
926-931,
1994[Abstract].
13.
Hain, J,
Nath S,
Mayrleitner M,
Fleischer S,
and
Schindler H.
Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.
Biophys J
67:
1823-1833,
1994[Abstract].
14.
Jideama, NM,
Noland TA,
Raynor RL,
Blobe GC,
Fabbro D,
Kazanietz MG,
Blumberg PM,
Hannun YA,
and
Kuo JF.
Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties.
J Biol Chem
271:
23277-23283,
1996
15.
MacLeod, KT,
and
Harding SE.
Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.
J Physiol
444:
481-498,
1991[Abstract].
16.
Mayrleitner, M,
Chandler R,
Schindler H,
and
Fleischer S.
Phosphorylation with protein kinases modulates calcium loading of terminal cisternae of sarcoplasmic reticulum from skeletal muscle.
Cell Calcium
18:
197-206,
1995[ISI][Medline].
17.
Moss, RL.
Ca2+ regulation of mechanical properties of striated muscle: mechanistic studies using extraction and replacement of regulatory proteins.
Circ Res
70:
865-884,
1992[Abstract].
18.
Noland, TA, Jr,
and
Kuo JF.
Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca2+-stimulated actomyosin MgATPase activity.
J Biol Chem
266:
4974-4978,
1991
19.
Noland, TA, Jr,
Raynor RL,
Jideama NM,
Guo X,
Kazanietz MG,
Blumberg PM,
Solaro RJ,
and
Kuo JF.
Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants.
Biochemistry
35:
4923-4931,
1996[ISI][Medline].
20.
Puceat, M,
Hilal-Dandan R,
Strulovici B,
Brunton LL,
and
Brown JH.
Differential regulation of protein kinase C isoform in isolated neonatal and adult rat cardiomyocytes.
J Biol Chem
269:
16938-16944,
1994
21.
Rybin, VO,
and
Steinberg SF.
Protein kinase C isoform expression and regulation in the developing rat heart.
Circ Res
74:
299-309,
1994[Abstract].
22.
Solaro, RJ,
and
Briggs FN.
Estimating the functional capabilities of sarcoplasmic reticulum in cardiac muscle.
Circ Res
34:
531-540,
1974[ISI][Medline].
23.
Solaro, RJ,
and
Rarick HM.
Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments.
Circ Res
83:
471-480,
1998
24.
Steinberg, SF,
Goldberg M,
and
Rybin VO.
Protein kinase C isoform diversity in the heart.
J Mol Cell Cardiol
27:
141-153,
1995[ISI][Medline].
25.
Sugden, PH,
and
Clerk A.
Cellular mechanisms of cardiac hypertrophy.
J Mol Med
76:
725-746,
1998[ISI][Medline].
26.
Takeishi, Y,
Chu G,
Kirkpatrick DM,
Li Z,
Wakasaki H,
Kranias EG,
King GL,
and
Walsh RA.
In vivo phosphorylation of cardiac troponin I by protein kinase C2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse hearts.
J Clin Invest
102:
72-78,
1998
27.
Wakasaki, H,
Koya D,
Schoen FJ,
Hoit BD,
Jirousek MR,
Ways DK,
Walsh RA,
and
King GL.
Targeted overexpression of protein kinase C II isoform in myocardium causes cardiomyopathy.
Proc Natl Acad Sci USA
94:
9320-9325,
1997
28.
Watson, JE,
and
Karmazyn M.
Concentration-dependent effects of protein kinase C-activating and -nonactivating phorbol esters on myocardial contractility, coronary resistance, energy metabolism, prostacyclin synthesis, and ultrastructure in isolated rat hearts. Effect of amiloride.
Circ Res
69:
1114-1131,
1991[Abstract].
29.
Wattanapermpool, J,
Guo X,
and
Solaro RJ.
The unique amino-terminal peptide of cardiac troponin I regulates myofibrillar ATPase activity only when it is phosphorylated.
J Mol Cell Cardiol
27:
1383-1391,
1995[ISI][Medline].
30.
Winegrad, S.
Myosin binding protein C, a potential regulator of cardiac contractility.
Circ Res
86:
6-7,
2000
31.
Wolska, BM,
Keller RS,
Evans CC,
Palmiter KA,
Phillips RM,
Muthuchamy M,
Oehlenschlager J,
Wieczorek DF,
de Tombe PP,
and
Solaro RJ.
Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin.
Circ Res
84:
745-751,
1999
32.
Wolska, BM,
and
Solaro RJ.
Method for isolation of adult mouse cardiac myocytes for studies of contraction and microflurimetry.
Am J Physiol Heart Circ Physiol
271:
H1250-H1255,
1996
33.
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
34.
Zhang, S,
Hiraano Y,
and
Hiraaoka M.
Arginine vasopressin-induced potentiation of unitary L-type Ca2+ channel current in guinea pig ventricular myocytes.
Circ Res
76:
592-599,
1995
35.
Zhang, ZH,
Johnson JA,
Chen L,
El-Sherif N,
Mochly-Rosen D,
and
Boutjdir M.
C2 region-derived peptides of beta-protein kinase C regulate cardiac Ca2+ channels.
Circ Res
80:
720-729,
1997