© 1997 American Heart Association, Inc.
Articles |
Left Ventricular Stretch Stimulates Angiotensin II– Mediated Phosphatidylinositol Hydrolysis and Protein Kinase C 
Isoform Translocation in Adult Guinea Pig Hearts
From the Division of Cardiology, Cardiovascular Center, University of Cincinnati (Ohio).
Correspondence to Richard A. Walsh, MD, Division of Cardiology, University of Cincinnati Medical Center, 231 Bethesda Ave, ML 542, Cincinnati, OH 45267. E-mail WALSHRA{at}UCBEH.SAN.UC.EDU
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract Stretch of neonatal cardiomyocytes activates phospholipase C with production of inositol trisphosphate and diacylglycerol in part by formation of angiotensin II (Ang II). However, the response of this pathway to physical stimuli in the adult heart is poorly understood. Thus, in isovolumic perfused guinea pig hearts, we characterized stretch-mediated phosphatidylinositol (PI) hydrolysis and protein kinase C (PKC) isoform translocation using elevated diastolic pressure. Balloon dilatation (minimum diastolic pressure, 25 mm Hg) of the left ventricle (LV) stimulated PI hydrolysis. Pretreatment of stretched hearts with the specific angiotensin (AT1) receptor antagonist losartan abolished stretch-mediated accumulation of inositol phosphates. To examine PKC isoform expression and activation under these conditions, whole-heart extracts were examined by immunoblot analysis. Ang II translocated PKC
to the particulate fraction. 4ß-Phorbol
12-myristate 13-acetate but not an inactive congener
translocated PKC to the particulate fraction and produced a decrease
in myocardial contractile function. Mechanical stretch also
translocated PKC to the particulate fraction; however, this was
attenuated but not abolished by losartan. We conclude that in
the adult heart, LV dilatation produced stretch-mediated activation of
phospholipase C, which resulted in PI hydrolysis and PKC activation
in part by stimulation of the local renin angiotensin
system. In contrast to stretch-mediated inositol phosphate
accumulation, PKC translocation is not prevented by AT1
receptor blockade, indicating that this PKC isoform can be
activated in response to mechanical deformation by an Ang
II–independent mechanism in the adult myocardium.
Key Words: signal transduction • stretch • inositol phosphate • protein kinase C • angiotensin II
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiac hypertrophy is a process wherein an increase in mass occurs in response to augmented global or regional chamber work largely as a consequence of an increase in the size of terminally differentiated cardiomyocytes. It has been known for some time that stretch can lead to an increase in protein synthesis in cardiac muscle.1 The mechanism(s) by which this physical stimulus is converted to biochemical events that lead to a change in the cardiac phenotype is poorly understood.2 3
It has been shown that in neonatal cardiac myocytes mechanical
deformation can activate a number of intracellular second
messengers and in particular the inositol phosphate–PKC (phospholipase
C) pathway.4 5 6 Cardiac myocytes and fibroblasts possess G
protein–coupled AT1 receptors for Ang II that
activate multiple intracellular signaling pathways. After
binding of Ang II to its specific receptor, the signal is transduced by
a specific heterotrimeric G protein (Gq), which undergoes dissociation
to the GTP-bound G
q subunit. Gq subsequently activates
the effector enzyme phosphoinositide-specific
phospholipase Cß, which is bound to the cytoplasmic face
of the plasma membrane and hydrolyzes the plasma membrane lipid,
phosphatidyl inositol 4,5-bisphosphate. This process generates two
biologically active intracellular messengers, DAG and IP3.
DAG activates phospholipid-dependent PKC, a potent mediator of
transcriptional regulation and hypertrophy in a variety of
cell types, and IP3, which in part maintains cellular
Ca2+ homeostasis by releasing Ca2+ from
endoplasmic reticulum stores. Although the cardiomyocyte
sarcoplasmic reticulum contains IP3 receptors, the role of
this polyphosphate in cardiac excitation-contraction coupling is
uncertain.
PKC isoenzyme expression has been characterized in
cardiomyocytes,7 8 9 vascular smooth
muscle,10 11 endothelium,12
and platelets.13 Presently, 11 isoenzymes of PKC
have been identified, and their cDNAs have been cloned from different
tissues and cell lines. The isoenzymes are classified into three
subfamilies: conventional PKCs (, ß1/ß2,
and 
, Ca2+ dependent), novel PKCs (
, , 
, and
, Ca2+ independent), and atypical PKCs (
, 
/
,
and µ, which do not bind or respond to phorbol esters). Recent
observations suggest that PKC may participate in neonatal myocyte
growth in response to mechanical stretch, which stimulates cells to
hypertrophy.4
The tumor-promoting drug PMA can replace DAG as an activator of conventional and novel PKC isozymes and has been used to evaluate the role of PKC isozymes in cell functions.14 Activation of PKC isozymes by PMA or hormones that activate DAG causes translocation of PKC from the cytosol to a particulate cell fraction, where the kinase regulates the activity of a number of proteins by phosphorylation. It has been shown that phosphorylation of substrates by PKC modulates Ca2+ and other ion levels, produces inotropic and chronotropic effects, alters gene expression, and induces secretion of cardiac growth factors and hypertrophy.9
Whether and to what extent mechanical deformation activates the phospholipase C pathway in the adult LV is unknown. We designed the present study to test the hypothesis that pathophysiological levels of mechanical stretch of the adult guinea pig LV can activate this ubiquitous bifurcated cell signaling pathway by an Ang II–dependent mechanism.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chemicals
Myo-[1,2-3H]inositol (45.6 Ci/nmol) was obtained from Dupont/NEN. Ang II, PMA, and PDD were purchased from Sigma Chemical Co. Enalaprilat and losartan were obtained from Merck Pharmaceutical Co.
Antibodies
Polyclonal anti-PKC antibodies, which recognize C-terminal amino
acids 313 to 326 of PKC, 726 to 737 of PKC, and 577 to 592 of
PKC, and respective inhibitory peptides were purchased
from Life Technologies. Alkaline phosphatase immunoblot
developing and chemiluminescence immunoblot developing
systems were from Bio-Rad. All other chemicals and reagents were
purchased from Fisher Scientific Corp.
Isolated Perfused Heart Preparation
Adult male Hartley strain guinea pigs (Charles River
Laboratories, Wilmington, Mass) were anesthetized with
intraperitoneal ketamine (54 mg/kg),
acepromazine maleate (1.8 mg/kg), and xylazine (10.9
mg/kg) and heparinized by injecting 200 U heparin sodium (1000
U/mL) into the abdominal aorta. Beating hearts were quickly excised,
weighed, and then perfused using a modified Langendorff preparation
with the ascending aorta terminally cannulated as previously
described.15 16 Briefly, hearts were perfused at a
constant flow rate of 10 mL/g per minute with oxygenated
Krebs-Henseleit buffer containing (mmol/L) NaCl 113.8, KCl 4.7,
MgSO4 · 7H2O 1.10,
KH2PO4 0.12, NaHCO3 23.6,
CaCl2 2.5, mannitol 6.0, and glucose 11.0, pH 7.4 to 7.5 at
37°C. A water-filled latex balloon attached to the end of a 3F Millar
high-fidelity micromanometer catheter (Millar
Instruments) was inserted into the LV through the mitral valve orifice
for pressure measurements and to induce LV stretch. All hearts were
paced at a constant heart rate of 250 to 300 bpm. The right ventricle
was vented, and the LV balloon was inflated sufficiently to obtain an
initial end-diastolic pressure of 3 to 5 mm Hg and
was isovolumic during initial perfusion.
Cardiac Mechanics
LV pressure and heart rate were continuously monitored on a
multichannel recorder (MK 200A, Gould) interfaced to an IBM
computer. Analog signals were digitized at a sampling frequency of 1000
Hz, and hemodynamic parameters were derived
from software developed in our laboratory.15 Fifteen to 20
cardiac cycles were averaged from each condition, and premature
contractions were excluded from analysis. LV developed pressure
(LV maximum-LV minimum) was measured, and the maximal rate of
isovolumic pressure development (maximum dP/dt) was derived and used as
an index of LV contractility; minimum dP/dt was chosen
as an indicator of the rate of LV isovolumic relaxation.
Phosphatidylinositol Hydrolysis in Response to Ang II
and Mechanical Stretch
After acquisition of baseline cardiac mechanics, a fixed volume
of recirculated LV perfusate (150 mL) was supplemented with
0.75 µCi/mL [3H]myoinositol for 2 hours to label
phospholipids, and the LV was perfused. Lithium chloride (5
mmol/L) was added 10 minutes before the addition of Ang II
or LV stretch to inhibit dephosphorylation of inositol
phosphates. In studies in which the effects of Ang II on
phosphoinositide hydrolysis were assessed, Ang II was
added in the indicated concentrations (0.1 to 10 µmol/L)
to the perfusate and circulated for 30 minutes. In experiments
examining the effect of LV stretch, the LV balloon was inflated to
achieve a minimum diastolic pressure of 25 mm Hg for
30 minutes. In some experiments, the AT1 receptor
antagonist losartan (1 µmol/L) or the
ACE inhibitor enalaprilat (1 µmol/L) was
added to the perfusate 10 minutes before mechanical stretch.
After completion of the studies, whole hearts were quickly frozen with
liquid nitrogen–cooled Wollenberger clamps and stored at -70°C
until analysis of inositol phosphates or PKC. For measurement
of inositol phosphates, frozen hearts were pulverized and
homogenized in 4 mL of 10% trichloroacetic acid using a
Tekmar tissue homogenizer. Water-soluble
3H-labeled products were extracted with water-saturated
ether (vol/vol) twice, with chloroform (vol/vol) once,
and then again with water-saturated ether (vol/vol). The labeled
inositol phosphates were resolved by ion-exchange HPLC using a Whatman
Partisil SAX column and a linear gradient of 0 to 1 mol/L
ammonium formate as previously described.17 Inositol
phosphates were identified by elution profiles compared with authentic
standards. AMP, ADP, and ATP served as internal markers. Inositol
phosphates were quantified by integrating the area under their
respective peaks (Fig 1
). Since isotopic
equilibrium may not have been reached by the 2- to 3-hour perfusion
period, the data are corrected for heart weight, and incorporation of
myoinositol was assessed by counting radioactivity in the whole-heart
homogenates before extraction. The results
represent total inositol phosphate accumulation (IP,
IP2, and IP3).
|
Immunoblot Analysis of PKC Isoform
Translocation
Membrane and cytosolic fractions of detergent-extracted PKC were
prepared according to methods previously described.18 19
Briefly, each whole guinea pig heart was pulverized, and the powdered
tissue from each heart was homogenized in 7 mL ice-cold
lysis buffer composed of (mmol/L) Tris-HCl 25, EGTA 5, EDTA 2,
and NaF 100 (pH 7.4) containing (µmol/L) leupeptin 20, E64 10,
pepstatin 120, and phenylmethylsulfonyl fluoride 200, plus
5 mmol/L dithiothreitol. An 800g crude
particulate fraction was discarded, and the supernatant was
centrifuged at 100 000g for 60 minutes. The pellet
constituted the membrane-particulate fraction, and the particulate-free
supernatant constituted the cytosolic fraction. Particulate fractions
were resuspended in homogenizing buffer containing
0.5% Triton X-100 and centrifuged at 100 000g for
60 minutes, and the resulting detergent-treated supernatant was the
membrane fraction. Protein content was estimated by a modification of
the method of Lowry (see Kiss et al15 ).
Cytosolic and membrane proteins were analyzed for PKC isoform
content by one-dimensional electrophoresis on 10%
SDS-polyacrylamide gels by a modification of the method of
Laemmli (see Kiss et al15 ). Gels were electrophoretically
transferred to Immunlite blotting membranes, blocked with 5% nonfat
milk in Tris-buffered saline at room temperature, and incubated
overnight with primary antibodies against specific PKC isoforms. Bound
antibody was detected by enhanced chemiluminescence. Quantification of
PKC, PKC, and PKC translocation was performed by laser
densitometric analysis of autoradiographs.
Calculations
The amount of loading of tritiated myoinositol in each heart was
quantified by determining the total amount of 3H (cpm) and
dividing by the heart weight (g) and radioactivity in the whole-heart
homogenate. PKC translocation was calculated by comparing
the relative densities of PKC isoform immunoblots in
particulate and cytosolic fractions in arbitrary units.
Statistical Analysis
All data are presented as mean±SEM. Inositol phosphate
accumulation, cardiac mechanics, and PKC translocation were directly
compared between control and stretched LV groups by Student's unpaired
t test. One- or two-way ANOVAs were used as appropriate for
multiple comparisons, followed by Dunnett's test for post hoc
analyses. Significance was accepted at P<.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Morphometric and Cardiac Mechanics
As shown in Table 1
, There
were no significant differences in LV weight–to–body weight or lung
weight–to–body weight ratios among the treatment groups. Likewise,
isovolumic LV mechanical parameters at a
physiological LV minimum pressure (5 mm Hg)
were similar among the groups.
|
Effects of Ang II on Inositol Phosphate Accumulation in
Isolated Perfused Guinea Pig Hearts
In an attempt to determine the effects of angiotensin
on the adult guinea pig myocardium, we perfused hearts with
0, 0.1, 1, and 10 µmol/L Ang II. Control hearts showed
little accumulation of inositol phosphates over the 30-minute study
period. In contrast, Ang II increased inositol phosphate accumulation
in a dose-related fashion (Fig 2).
|
.05 vs
control.
Effects of LV Stretch on Inositol Phosphate
Accumulation
Fig 1
demonstrates representative
chromatographs from an LV homogenate in the absence
of stretch or drug intervention compared with a heart in which the LV
was stretched at 25 mm Hg for 30 minutes. Hearts that underwent
stretch (Fig 3) manifested a 256%
increase in inositol phosphate accumulation relative to nonstretched
hearts (3510±1007 versus 8991±2332 cpm/g heart wt for nonstretched
versus stretched hearts, respectively; P<.05). Ang II
(10 µmol/L) stimulation similarly augmented inositol
phosphate accumulation (6176±1068 versus 8991±2332 cpm/g heart wt for
nonstretched versus stretched hearts, respectively).
|
Effects of AT1 Receptor Blockade and ACE Inhibition on
Stretch-Mediated Inositol Phosphate Accumulation
Ang II has been implicated as a critical mediator of the
stretch-induced hypertrophic responses in neonatal cardiac
myocytes.20 In an attempt to elucidate the mechanism of
stretch-induced inositol phosphate accumulation, the effects of the
specific AT1 Ang II receptor antagonist
losartan were explored. Losartan (1
µmol/L) effectively inhibited the stretch-mediated inositol
phosphate accumulation (Fig 3). Inositol phosphate accumulation was
significantly lower in the losartan plus stretch group than in
the stretched group (losartan plus stretch, 703±164 cpm/g
heart wt; stretch, 8891±2332 cpm/g heart wt; P<.05).
Inositol phosphate accumulation also was lower with AT1
blockade (losartan plus stretch, 703±164; no stretch,
3510±1007 cpm/g heart wt; P<.05) (Fig 3). However,
addition of losartan to nonstretched buffer-perfused hearts
(n=3) failed to lower inositol phosphate levels (3411±227 cpm/g heart
wt) below those of unstretched hearts (3510±1007 cpm/g heart wt, n=7
hearts) in the absence of AT1 blockade. Therefore, it does
not appear that Ang II generation occurred in the unstretched heart
under these experimental conditions.
We have previously demonstrated ACE-independent generation of Ang II in
the primate myocardium.21 In order to
determine whether and to what extent ACE-independent generation of Ang
II contributes to stretch-mediated phosphatidylinositol hydrolysis, we
measured inositol phosphate accumulation in the presence of the ACE
inhibitor enalaprilat. Enalaprilat (1 µmol/L)
also completely inhibited stretch-mediated inositol phosphate
accumulation (Fig 3).
PKC Isoforms in Adult Guinea Pig Heart: Effects of Phorbol Ester
and Hormonal Stimulation
Using Western blot analyses, we detected , , and
isoforms of PKC in lysates prepared from adult hearts. The bands
detected by immunoblotting had molecular masses of
80, 96, and 78 kD, corresponding to those of PKC isoforms , ,
and , respectively. Antibodies directed against the ß, , and
isoforms of PKC did not show immunoreactivity (data not shown). In
subsequent experiments, we assessed the effects of Ang II and PMA by
their ability to increase membrane-associated immunoreactivity for each
of these PKC isoforms.4 PMA treatment increased the amount
of immunoreactivity in PKC and PKC, but not in PKC, in
membranes of adult hearts.
PMA (100 nmol/L) stimulation depressed isovolumic LV
mechanics compared with baseline values (Fig 4). There was a 67% decrease in
developed pressure during PMA stimulation (baseline, 118±4
mm Hg; PMA, 39±5 mm Hg; P<.05). Similarly, there
were 62% and 63% decreases in the rates of
contractility and relaxation (+dP/dt: baseline,
2336±51 mm Hg/s; PMA, 893±51 mm Hg/s;
-dP/dt: baseline, -1924±70 mm Hg/s; PMA, -720±50
mm Hg/s; each, P<.05). Stimulation with inactive
phorbol ester PDD had no effect on baseline isovolumic LV
mechanics.
|
In order to determine the effects of Ang II stimulation on PKC
activation, we measured PKC translocation from the cytosol to the
membrane. Adult hearts were perfused with 10 µmol/L Ang
II for 30 minutes after equilibration on a modified Langendorff
preparation. PKC and PKC showed no translocation to the membrane
with Ang II stimulation. In contrast, PKC translocated with Ang II
stimulation (Fig 5).
|
Effects of Stretch on Protein Kinase Translocation
Additional hearts were studied to examine the effects of LV
stretch on PKC translocation. The hearts were excised and placed on a
modified Langendorff preparation. The LV was preloaded with 25
mm Hg minimum diastolic pressure after a 30-minute
equilibration period. A time course of 0, 2.5, 5, 7.5, 10, and 20
minutes showed maximal translocation at 7.5 minutes, which was still
detectable at 20 minutes (Fig 6). Whole
hearts that were nonstretched (n=5) and stretched (n=5) for 7.5 minutes
at 25 mm Hg were perfused, extracted, and probed with PKC,
PKC, and PKC antibodies. PKC translocation in the stretched
hearts increased 5.5-fold from cytosol to membrane over baseline values
(from 0.304±0.123 to 1.681±0.475 arbitrary units, P<.05).
Neither PKC (from 0.265±0.031 to 0.548±0.213 arbitrary units) nor
PKC underwent significant translocation (Table 2).
|
|
Effects of AT1 Receptor Blockade on PKC
Translocation
In order to elucidate the potential role of Ang II on
stretch-mediated PKC translocation, 1 µmol/L
losartan was added to the perfusate, and the LV balloon
was inflated to 25 mm Hg minimum diastolic pressure
for 7.5 minutes. In contrast to the effect on phosphatidylinositol
hydrolyses, the same degree of AT1 receptor blockade with
losartan attenuated but did not abolish stretch-induced PKC
translocation. There was a 3.5-fold increase in PKC translocation
compared with baseline (1.05±0.201 versus 0.304±0.123 arbitrary
units, P<.05) (Fig 7) (Table 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The principal result of the present study is that pathophysiological distension of the adult LV activates the phospholipase C signal transduction pathway. The key observations include the following: (1) Ang II, phorbol ester stimulation, and mechanical deformation stimulate phosphatidylinositol hydrolysis and translocate PKC
in LV myocardium. (2)
AT1 receptor inhibition (losartan) and ACE
inhibition (enalaprilat) completely blocked inositol phosphate
hydrolysis but not PKC translocation; to our knowledge, these are the
first data in the adult heart that demonstrate ex vivo localized
production of Ang II and resultant coupling to downstream
signal transduction. (3) PKC activation by phorbol ester stimulation
produced a negative inotropic effect in the isovolumically contracting
guinea pig LV. A variety of cell-signaling pathways have been demonstrated to be activated by cardiovascular mechanical deformation.22 These include stretch-activated ion channels, an extracellular matrix protein/integrin–linked pathway, the Na+-H+ antiporter, phospholipases C, D, and A2, protein kinase C, tyrosine kinases, P21ras, mitogen-activated protein kinases, and 90-kD S6 kinase. This variety of signal transduction pathways and the potential cross talk among them has largely been studied in cultured neonatal rat cardiomyocytes and avian myocytes.23 The determinants of mechanotransduction in the adult heart have been more difficult to study because of the inability to passage terminally differentiated myocytes in culture. As a corollary, there are few data regarding the existence and importance of various stretch-activated signal transduction pathways in the adult LV, despite the possibility that these pathways may be importantly affected by developmental regulation and the multicellular environment of the whole organ.
In particular, Sadoshima et al5 have demonstrated the importance of Ang II in the stretch-induced production of the hypertrophy phenotype in the cultured neonatal cardiomyocyte. They demonstrated that the addition of angiotensin under these conditions increased protein synthesis and induced immediate-early genes and growth factors and that AT1, but not AT2, receptor blockade prevented these actions.20 Static stretch of neonatal cardiocytes recapitulated these events, whereas stretch-induced hypertrophy was prevented by AT1 receptor blockade. Immunoelectron microscopy suggested that this was mediated in part by an autocrine action of Ang II.20 The present study extends these observations to the adult LV. We have demonstrated that pathophysiological distension (25 mm Hg) of the isolated isovolumically contracting LV activates phospholipase C–mediated accumulation of inositol phosphates and activation of PKC. AT1 receptor blockade completely inhibited IP3 accumulation but not PKC translocation. In this regard, it is interesting that chelation of intracellular Ca2+, but not downregulation of PKC, suppressed Ang II–mediated activation of mitogen-activated protein kinase and 90-kD S6 kinase.24 These important downstream components of tyrosine kinase signaling pathways may be critically affected by stretch, Ang II–stimulated accumulation of IP3, and the resultant modulation of intramyocyte Ca2+ stores. Taken together, the results of the present study demonstrate autocrine and/or paracrine production of Ang II by the adult heart and the importance of the local renin-angiotensin system in cardiac mechanotransduction.
Komuro et al4 have also used cultured neonatal
cardiomyocytes to examine the role of stretch-induced Ang
II–mediated activation of the phospholipase C signal transduction
pathway. Static stretch of these myocytes to 20% of resting length
(corresponding to a 1.73 increase in chamber volume [ie,
1.23]) increased inositol phosphate levels, whereas the
accumulation of c-fos was attenuated by PKC inhibition. In
the present study, we demonstrate that LV distension in the adult
heart to levels seen in pathological states directly induces PKC
translocation. The incomplete inhibition of PKC activation by
AT1 receptor blockade suggests the presence of an Ang
II–independent process in mature myocardium. It is
possible that other Gq-coupled receptors, such as the endothelin or the
1-adrenergic receptor, are activated by stretch
in the adult heart. Alternatively, stretch may stimulate phospholipase
D–mediated hydrolysis of phosphatidylcholine. The resultant formation
of phosphatidic acid and its metabolism via a
phosphohydrolase to DAG may activate PKC.25 The
cellular origin of putative signaling peptides cannot be deduced from
the present study. However, the isolated isovolumic heart
preparation permits the study of these processes without the
confounding effects of neurally modulated or circulating hormonal
factors and establishes their presence and importance at the
whole-organ level.
We immunologically identified three major isoforms of PKC present
in the adult guinea pig whole-heart homogenates.
Whole-heart preparations expressed , , and isoforms, with the
Ca2+-independent isoform being the most immunoreactive.
Others have shown that multiple PKC isoforms are expressed in the rat
heart in an age-dependent fashion.26 PKC, PKC,
PKC, and PKC were detected in whole extracts from neonatal
ventricle and cultured neonatal ventricular myocytes.
However, only two PKC isoforms (PKC and PKC) were detected in
total protein extracts from isolated adult ventricular
myocytes.9 We detected small amounts of PKC and large
amounts of PKC in total protein extracts from whole adult guinea pig
hearts. This finding most likely represents PKC and PKC
derived from nonmuscle cells in the heart or from the atria, since
whole-heart lysates were used in the present study.
It has recently been demonstrated that AT-1 cells (a transplantable
tumor lineage cell line derived from transgenic mouse atrial
cardiomyocytes) express PKC, PKC, and PKC
isoforms27 ). Stimulation with endothelin induces selective
membrane association of only the Ca2+-independent PKC;
translocation of PKC was not detected.27 Our findings
are consistent with the hypothesis that in cardiac myocytes,
receptors coupled to phospholipase C–mediated
phosphoinositide hydrolysis selectively induce the
translocation of PKC (but not PKC) to the particulate fraction,
whereas in noncardiac myocytes receptors coupled to
phosphatidylinositide hydrolysis increase the membrane association of
both PKC and PKC isoforms. It should be emphasized that PKC,
PKC, and PKC comigrated at their expected molecular weights and
that affinity-purified isoform-specific antibody binding was either
blocked or attenuated by inhibitory peptides. We were
unable to precisely quantify the reciprocal decrease of PKC in the
soluble fraction and increase in the particulate fraction because of
variations in the background of the gels among experiments.
The effect of phorbol esters to translocate PKC from the soluble to the
particulate fraction has been used as an indicator of PKC
activation.28 Thirty-minute stimulation of the heart with
PMA translocated PKC to the particulate fraction. A number of
laboratories have reported that phorbol esters modulate contractile
function of neonatal and adult rat cardiomyocytes and
perfused hearts.18 29 30 In the present study, we have
shown that PMA produces significant negative inotropy in the isolated
isovolumic buffer-perfused guinea pig LV. There was a 67% decrease in
developed pressure and equivalent decreases in the rates of contraction
and relaxation (62% and 63% reduction, respectively). There are
several mechanisms by which phorbol ester stimulation of PKC may
contribute to myocyte contractile depression. There is some evidence
that PMA stimulation causes a decrease in the Ca2+
transient in adult murine cardiomyocytes.30 In
embryonic chicken dorsal root ganglion neurons, both a short-acting DAG
analogue and a phorbol ester decrease the Ca2+
current.23 Since phorbol esters induce translocation of
certain PKC isoforms to the myofilaments,31 some
investigators have focused on direct phosphorylation of
proteins within the contractile machinery as a mechanism for
PKC-dependent modulation of contractile function. Cardiac troponins I
and C have been identified as endogenous substrates for
phosphorylation by PKC in the heart.32
Phosphorylization of troponin I leads to a decrease in myofilament
sensitivity to Ca2+ and to a reduction in myofibrillar
actin myosin ATPase activity and could contribute to a negative
inotropic response. Troponin T has been shown to be a target for
phorbol ester–induced phosphorylation in neonatal, but
not adult, ventricular myocytes.33 The
functional role of C-protein phosphorylation is not
known. Finally, there is recent evidence that PKC
phosphorylates myosin light chain 2.32 34
Whether PKC-mediated phosphorylation of myosin light
chain 2 alters myofibrillar Ca2+ sensitivity is unclear. It
is possible that enhanced constitutive19 and/or
stretch-activated phospholipase C hydrolysis may contribute to
altered function of the hypertrophied and failing adult heart.
Experiments are under way in our laboratories to directly address this
issue by cardiac-specific overexpression of Gq in genetically
engineered mice.
In summary, we have demonstrated that mechanical distension of
the adult LV resulted in inositol phosphate accumulation and
isoform-specific translocation of PKC from the soluble to the
particulate fraction in guinea pig whole-heart lysates. Inositol
phosphate accumulation but not translocation of PKC appears to be
completely mediated by Ang II–coupled Gq-linked phospholipase C
hydrolysis. Taken together, the results of the present study
suggest that stretch-induced activation of the phospholipase C signal
transduction pathway during pathological states may play a critical
role in modulating growth and contractile function in the intact
LV.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported in part by National Institutes of Health grants SCOR in Heart Failure (P50 HL-52318, Dr Walsh), HL-33579 (Dr Walsh), and HL-49267 (Dr Dorn) and by the Veterans Administration. Dr Dorn is an Established Investigator of the American Heart Association.
Received October 25, 1996; accepted July 5, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Morgan HE, Baker KM. Cardiac hypertrophy: mechanical, neural, and endocrine dependence. Circulation. 1991;83:13-25.
2.
Cooper G IV, Mercer WE, Hoober JK, Gordon PR, Kent RL,
Laura IK, Marino TA. Load regulation of the properties of adult
feline cardiocytes: the role of substrate adhesion.
Circ Res. 1986;58:692-705.
3. Komuro I, Yazaki Y. Control of cardiac gene expression by mechanical stress. Annu Rev Physiol. 1993;55:55-75.[Medline] [Order article via Infotrieve]
4.
Komuro I, Katoh Y, Kaida T, Shibazaki Y, Kurabayashi
M, Hoh E, Takaku F, Yazaki Y. Mechanical loading stimulates cell
hypertrophy and specific gene expression in cultured rat
cardiac myocytes: possible role of protein kinase C activation.
J Biol Chem. 1991;266:1265-1268.
5.
Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo
S. Molecular characterization of the stretch-induced adaptation
of cultured cardiac cells: an in vitro model of
load-induced cardiac hypertrophy. J Biol
Chem. 1992;267:10551-10560.
6. Sugden PH, Bogoyevitch MA. Intracellular signalling through protein kinases in the heart. Cardiovasc Res. 1995;30:478-492.[Medline] [Order article via Infotrieve]
7.
Bogoyevitch MA, Parker PJ, Sugden PH.
Characterization of protein kinase C isotype expression in adult rat
heart: protein kinase C- is a major isotype present, and it is
activated by phorbol esters, epinephrine, and
endothelin. Circ Res. 1993;72:757-767.
8.
Pucéat M, Hilal-Dandan R, Strulovici B, Brunton
LL, Brown JH. Differential regulation of protein kinase C
isoforms in isolated neonatal and adult rat
cardiomyocytes. J Biol Chem. 1994;269:16938-16944.
9. Steinberg SF, Goldberg M, Rybin VO. Protein kinase C isoform diversity in the heart. J Mol Cell Cardiol. 1995;27:141-153.[Medline] [Order article via Infotrieve]
10. Khalil RA, Lajoie C, Resnick MS, Morgan KG. Ca2+-independent isoforms of protein kinase C differentially translocate in smooth muscle. Am J Physiol. 1992;262(Heart Circ Physiol. 32):C714-C719.
11. Liou YM, Morgan KG. Redistribution of protein kinase C isoforms in association with vascular hypertrophy of rat aorta. Am J Physiol. 1994;267(Cell Physiol. 36):C980-C989.
12.
Bussolino F, Silvagno F, Garbarino G, Costamagna C,
Sanavio F, Arese M, Soldi R, Aglietta M, Pescarmona G, Cam'ssi G,
Bosia A. Human endothelial cells are targets for
platelet-activating factor (PAF): activation of and ß protein
kinase C isozymes in endothelial cells stimulated by
PAF. J Biol Chem. 1994;269:2877-2886.
13. Harrington EO, Ware JA. Diversity of the protein kinase C gene family: implications for cardiovascular disease. Trends Cardiovasc Med. 1995;5:193-199.
14. Kraft AS, Anderson WB. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1993;301:621-623.
15.
Kiss E, Ball NA, Kranias EG, Walsh RA.
Differential changes in cardiac phospholamban and sarcoplasmic
reticular Ca2+-ATPase protein levels: effects on
Ca2+ transport and mechanics in compensated
pressure-overload hypertrophy and congestive heart
failure. Circ Res. 1995;77:759-764.
16. Collins JF, Pawloski-Dahm C, Davis MG, Ball N, Dorn GW II, Walsh RA. The role of the cytoskeleton in left ventricular pressure overload hypertrophy and failure. J Mol Cell Cardiol. 1996;28:1435-1443.[Medline] [Order article via Infotrieve]
17.
Dorn GW II, Becker MW. Thromboxane
A2 stimulated signal transduction in vascular smooth
muscle. J Pharmacol Exp Ther. 1993;265:447-456.
18. Yuan S, Sunahara FA, Sen AK. Tumor-promoting phorbol esters inhibit cardiac functions and induce redistribution of protein kinase C in perfused beating rat heart. Circ Res. 1987;61:373-378.
19.
Gu X, Bishop SP. Increased protein kinase C and
isozyme redistribution in pressure-overload cardiac
hypertrophy in the rat. Circ Res. 1994;75:926-931.
20. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977-984.[Medline] [Order article via Infotrieve]
21. Hoit BD, Shao Y, Kinoshita A, Gabel M, Husain A, Walsh RA. Effects of angiotensin II generated by an angiotensin converting enzyme-independent pathway on left ventricular performance in the conscious baboon. J Clin Invest. 1995;95:1519-1527.
22. Watson PA. Mechanical activation of signaling pathways in the cardiovascular system. Trends Cardiovasc Med. 1996;6:73-79.
23. Leatherman GF, Kim D, Smith TW. Effect of phorbol esters on contractile state and calcium flux in cultured chick heart cells. Am J Physiol. 1987;253(Heart Circ Physiol. 22):H205-H209.
24.
Sadoshima J, Qiu Z, Morgan JP, Izumo S.
Angiotensin II and other hypertrophic stimuli mediated by G
protein–coupled receptors activate tyrosine kinase,
mitogen-activated protein kinase, and 90-kD S6 kinase in
cardiac myocytes: the critical role of Ca2+-dependent
signaling. Circ Res. 1995;76:1-15.
25. Exton JH. Phosphatidylcholine breakdown and signal transduction. Biochim Biophys Acta. 1994;1212:26-42.[Medline] [Order article via Infotrieve]
26.
Rybin VO, Steinberg SF. Protein kinase C isoform
expression and regulation in the developing rat heart.
Circ Res. 1994;74:299-309.
27.
Jiang T, Pak E, Zhang H, Kline RP, Steinberg SF.
Endothelin-dependent actions in cultured AT-1 cardiac myocytes: the
role of the isoform of protein kinase C. Circ
Res. 1996;78:724-736.
28. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988;334:661-665.[Medline] [Order article via Infotrieve]
29.
Dösemeci A, Dhallan RS, Cohen NM, Lederer WJ,
Rogers TB. Phorbol ester increases calcium current and simulates
the effects of angiotensin II on cultured neonatal rat
heart myocytes. Circ Res. 1988;62:347-357.
30.
Capogrossi MC, Kaku T, Filburn CR, Pelto DJ, Hansford
RG, Spurgeon HA, 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. 1990;66:1143-1155.
31. Disatnik MH, Buraggi G, Mochly-Rosen D. Localization of protein kinase C isozymes in cardiac myocytes. Exp Cell Res. 1994;210:287-297.[Medline] [Order article via Infotrieve]
32.
Venema RC, Kuo JF. Protein kinase C-mediated
phosphorylation of troponin I and C-protein in isolated
myocardial cells is associated with inhibition of myofibrillar
actomyosin MgATPase. J Biol Chem. 1993;268:2705-2711.
33.
Robertson SP, Johnson JD, Holroyde MJ, Kranias EG,
Potter JD, Solaro RJ. The effect of troponin I
phosphorylation on the Ca2+-binding
properties of the Ca2+-regulatory site of bovine cardiac
troponin. J Biol Chem. 1982;257:260-263.
34. Venema RC, Raynor RL, Noland TA, Kuo JF. Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2. Biochem J. 1993;294:401-406.
This article has been cited by other articles:
 |
|
 |
|
  C. N. White, G. A. Figtree, C.-C. Liu, A. Garcia, E. J. Hamilton, K. K. M. Chia, and H. H. Rasmussen Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase Am J Physiol Cell Physiol, April 1, 2009; 296(4): C693 - C700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bupha-Intr, K. M. Haizlip, and P. M. L. Janssen Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro Am J Physiol Heart Circ Physiol, March 1, 2009; 296(3): H806 - H814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Vincent, N. Duquesnes, C. Christov, T. Damy, J.-L. Samuel, and B. Crozatier Dual level of interactions between calcineurin and PKC-{varepsilon} in cardiomyocyte stretch Cardiovasc Res, July 1, 2006; 71(1): 97 - 107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kang and J. W. Walker Endothelin-1 and PKC Induce Positive Inotropy Without Affecting pHi in Ventricular Myocytes. Experimental Biology and Medicine, June 1, 2006; 231(6): 865 - 870. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Mozaffari, C. Patel, and S. W. Schaffer Mechanisms Underlying Afterload-Induced Exacerbation of Myocardial Infarct Size: Role of T-Type Ca2+ Channel Hypertension, May 1, 2006; 47(5): 912 - 919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hoshijima Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1313 - H1325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Zankov, M. Omatsu-Kanbe, T. Isono, F. Toyoda, W.-G. Ding, H. Matsuura, and M. Horie Angiotensin II Potentiates the Slow Component of Delayed Rectifier K+ Current via the AT1 Receptor in Guinea Pig Atrial Myocytes Circulation, March 14, 2006; 113(10): 1278 - 1286. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Cho, D. Lee, S. H. Lee, and W.-K. Ho Receptor-induced depletion of phosphatidylinositol 4,5-bisphosphate inhibits inwardly rectifying K+ channels in a receptor-specific manner PNAS, March 22, 2005; 102(12): 4643 - 4648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Browe and C. M. Baumgarten Angiotensin II (AT1) Receptors and NADPH Oxidase Regulate Cl- Current Elicited by {beta}1 Integrin Stretch in Rabbit Ventricular Myocytes J. Gen. Physiol., August 30, 2004; 124(3): 273 - 287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. B. Roman, P. H. Goldspink, E. Spaite, D. Urboniene, R. McKinney, D. L. Geenen, R. J. Solaro, and P. M. Buttrick Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2089 - H2095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Takahashi, T. Anzai, T. Yoshikawa, Y. Maekawa, K. Mahara, M. Iwata, H. K. Hammond, and S. Ogawa Angiotensin receptor blockade improves myocardial beta-adrenergic receptor signaling in postinfarction left ventricular remodeling: A possible link between beta-adrenergic receptor kinase-1 and protein kinase C epsilon isoform J. Am. Coll. Cardiol., January 7, 2004; 43(1): 125 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wang, X. Liu, E. Sentex, N. Takeda, and N. S. Dhalla Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2277 - H2287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Brutsaert Cardiac Endothelial-Myocardial Signaling: Its Role in Cardiac Growth, Contractile Performance, and Rhythmicity Physiol Rev, January 1, 2003; 83(1): 59 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Buhagiar, P. S. Hansen, N. L. Bewick, and H. H. Rasmussen Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump Am J Physiol Cell Physiol, September 1, 2001; 281(3): C1059 - C1063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Pass, Y. Zheng, W. B. Wead, J. Zhang, R. C. X. Li, R. Bolli, and P. Ping PKC{epsilon} activation induces dichotomous cardiac phenotypes and modulates PKC{epsilon}-RACK interactions and RACK expression Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H946 - H955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-G. Shin, J. V. Barnett, P. Chang, S. Reddy, D. C. Drinkwater, R. N. Pierson, R. G. Wiley, and K. T. Murray Molecular heterogeneity of protein kinase C expression in human ventricle Cardiovasc Res, November 1, 2000; 48(2): 285 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Takeishi, P. Ping, R. Bolli, D. L. Kirkpatrick, B. D. Hoit, and R. A. Walsh Transgenic Overexpression of Constitutively Active Protein Kinase C {epsilon} Causes Concentric Cardiac Hypertrophy Circ. Res., June 23, 2000; 86(12): 1218 - 1223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. De Windt, H. W. Lim, S. Haq, T. Force, and J. D. Molkentin Calcineurin Promotes Protein Kinase C and c-Jun NH2-terminal Kinase Activation in the Heart. CROSS-TALK BETWEEN CARDIAC HYPERTROPHIC SIGNALING PATHWAYS J. Biol. Chem., April 28, 2000; 275(18): 13571 - 13579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. W. Dorn II, N. M. Tepe, G. Wu, A. Yatani, and S. B. Liggett Mechanisms of Impaired beta -Adrenergic Receptor Signaling in Galpha q-Mediated Cardiac Hypertrophy and Ventricular Dysfunction Mol. Pharmacol., February 1, 2000; 57(2): 278 - 287. [Abstract] [Full Text]
|
|
|
|
|
|
T. Jalili, Y. Takeishi, G. Song, N. A. Ball, G. Howles, and R. A. Walsh PKC translocation without changes in Galpha q and PLC-beta protein abundance in cardiac hypertrophy and failure Am J Physiol Heart Circ Physiol, December 1, 1999; 277(6): H2298 - H2304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Kim, T. Lee, J. Fu, and M. E. Ritchie Characterization of MAP kinase and PKC isoform and effect of ACE inhibition in hypertrophy in vivo Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1808 - H1816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Dostal and K. M. Baker The Cardiac Renin-Angiotensin System : Conceptual, or a Regulator of Cardiac Function? Circ. Res., October 1, 1999; 85(7): 643 - 650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Jalili, Y. Takeishi, and R. A Walsh Signal transduction during cardiac hypertrophy: the role of G{alpha}q, PLC {beta}I, and PKC Cardiovasc Res, October 1, 1999; 44(1): 5 - 9. [Full Text] [PDF]
|
|
|
|
|
|
Y. Takeishi, T. Jalili, N. A. Ball, and R. A. Walsh Responses of Cardiac Protein Kinase C Isoforms to Distinct Pathological Stimuli Are Differentially Regulated Circ. Res., August 6, 1999; 85(3): 264 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A.M van Kesteren, J. J Saris, D. H.W Dekkers, J. M.J Lamers, P. R Saxena, M. A.D.H Schalekamp, and A.H.J. Danser Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II Cardiovasc Res, July 1, 1999; 43(1): 148 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Walsh Calcineurin Inhibition as Therapy for Cardiac Hypertrophy and Heart Failure : Requiescat in Pace? Circ. Res., April 2, 1999; 84(6): 741 - 743. [Full Text] [PDF]
|
|
|
|
|
|
N. Bowling, R. A. Walsh, G. Song, T. Estridge, G. E. Sandusky, R. L. Fouts, K. Mintze, T. Pickard, R. Roden, M. R. Bristow, et al. Increased Protein Kinase C Activity and Expression of Ca2+-Sensitive Isoforms in the Failing Human Heart Circulation, January 26, 1999; 99(3): 384 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bartunek, E. O. Weinberg, M. Tajima, S. Rohrbach, and B. H. Lorell Angiotensin II Type 2 Receptor Blockade Amplifies the Early Signals of Cardiac Growth Response to Angiotensin II in Hypertrophied Hearts Circulation, January 12, 1999; 99(1): 22 - 25. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takeishi, A. Bhagwat, N. A. Ball, D. L. Kirkpatrick, M. Periasamy, and R. A. Walsh Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure Am J Physiol Heart Circ Physiol, January 1, 1999; 276(1): H53 - H62. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A Woodcock, S. J Matkovich, and O. Binah Ins(1,4,5)P3 and cardiac dysfunction Cardiovasc Res, November 1, 1998; 40(2): 251 - 256. [Full Text] [PDF]
|
|
|
|
|
|
H. E. Cingolani, B. V. Alvarez, I. L. Ennis, and M. C. Camilion de Hurtado Stretch-Induced Alkalinization of Feline Papillary Muscle : An Autocrine-Paracrine System Circ. Res., October 19, 1998; 83(8): 775 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Laser, V. S. Kasi, M. Hamawaki, G. Cooper IV, C. M. Kerr, and D. Kuppuswamy Differential Activation of p70 and p85 S6 Kinase Isoforms during Cardiac Hypertrophy in the Adult Mammal J. Biol. Chem., September 18, 1998; 273(38): 24610 - 24619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Middleton and R. D. Harvey PKC regulation of cardiac CFTR Cl- channel function in guinea pig ventricular myocytes Am J Physiol Cell Physiol, July 1, 1998; 275(1): C293 - C302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sil, V. Kandaswamy, and S. Sen Increased Protein Kinase C Activity in Myotrophin-Induced Myocyte Growth Circ. Res., June 15, 1998; 82(11): 1173 - 1188. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||