Download D ifferential regulation of cardiac protein kinase C isozyme

Cardiovascular Research 56 (2002) 52–63
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Differential regulation of cardiac protein kinase C isozyme expression
after aortic banding in rat
´ b , Steffen Schon
¨ a , Mathias M. Borst c , Ruth H. Strasser a , *
Martin U. Braun a , Paul LaRosee
a
Department of Cardiology, Medical Clinic II, University of Technology Dresden, Fetscherstr. 76, 01307 Dresden, Germany
b
Department of Cardiology, Medical Center, University of Mannheim, Mannheim, Germany
c
Department of Cardiology, Angiology and Pulmology, Medical Center, University of Heidelberg, Heidelberg, Germany
Received 16 November 2001; accepted 29 May 2002
Abstract
Objective: Protein kinase C (PKC) plays a key role in myocardial hypertrophy. To evaluate whether its isoforms are expressed
differentially during gradual development of pressure-overload-induced cardiac hypertrophy, banding of the ascending aorta was used as
an experimental model of left ventricular hypertrophy. Methods: One, 7 and 30 days after sham operation or aortic banding in male
Wistar rats, the PKC activity and the expression of the cardiac PKC isozymes (PKC-a, -d, -´ and -z), both at the protein and the mRNA
level, were determined in the left and right ventricle. Results: Left ventricular hypertrophy developed rapidly as early as 1 day after aortic
banding followed by further progression at day 7 and day 30. This was paralleled by an increased total PKC enzyme activity in the
cytosol fraction and a selectively enhanced protein expression of PKC-d (day 7, 267618%; day 30, 289612%) and PKC-a (day 7,
212620%; day 30, 193614%). The protein amount of PKC-´ was not changed in either group. This differential protein expression was
associated with a significant increase of the absolute mRNA levels for PKC-d and PKC-a up to 202620% (day 30) and 177617% (day
30), whereas significant alterations in the PKC-´ mRNA levels were not detected. A selective upregulation of PKC-a and PKC-d, both on
the protein and on the mRNA level, was also noted in the right ventricle during the development of right ventricular hypertrophy,
suggesting an adaptive response following elevated left ventricular enddiastolic pressure after long-term aortic banding for 30 days.
Conclusions: This study characterizes in the right and left ventricle a differential regulation of the dominant PKC isozymes in
pressure-overload cardiac hypertrophy both at the protein and the mRNA level.
 2002 Elsevier Science B.V. All rights reserved.
Keywords: Gene expression; Hypertrophy; Protein kinases; Signal transduction; Ventricular function
1. Introduction
The development of cardiac hypertrophy is associated
with an increased risk for cardiovascular events such as
sudden death and congestive heart failure [1]. Myocardial
hypertrophy due to systemic hypertension and / or aortic
stenosis is based on typical morphological and biochemical
changes of the cardiomyocytes, including an increased cell
size, an enhanced protein content [2], an induction of
immediate early genes such as c-fos and c-myc [3] and the
*Corresponding author. Tel.: 149-351-450-1700; fax: 149-351-4501702.
E-mail address: [email protected] (R.H. Strasser).
re-expression of fetal gene programmes [4]. However, little
is known about the molecular signaling mechanisms which
mediate these cellular features of the hypertrophic response.
Extracellular signals such as norepinephrine [5] and
angiotensin II [6] can initiate cardiac hypertrophy both in
vivo and in isolated cardiomyocytes. One of their common
features is the activation of protein kinase C (PKC), an
enzyme known to be involved in cell differentiation and
proliferation [7]. In addition, mechanical stress, which
represents one of the strongest hypertrophic stimuli, promotes an activation of different kinase cascades, including
Time for primary review 31 days.
0008-6363 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 02 )00511-4
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
c-jun kinases, mitogen-activated protein kinases (MAPKs)
and a pronounced activation of PKC [8]. Several in vivo
studies of volume-overload hypertrophy [9] and systemic
hypertension [10] have suggested that PKC plays a pivotal
role in the continuous development of cardiac hypertrophy.
Furthermore, direct stimulation of PKC with phorbolester
induces cellular hypertrophy in cultured isolated adult
cardiomyocytes [11]. Thus, quite distinct hypertrophic
stimuli converge at the level of PKC.
PKC consists of a family of at least 11 isozymes [12].
Based on their structural and regulatory properties these
PKC isozymes can be divided into three major groups: the
conventional, Ca 21 -dependent isoforms, including PKC-a,
PKC-b and PKC-g, the novel, Ca 21 -independent isoforms,
PKC-d and PKC-´, and the atypical isoforms, PKC-z and
PKC-l. The PKC isozymes are expressed differentially in
various organs and tissues [13]. In adult rat the dominant
PKC isoforms PKC-a, PKC-d and PKC-´ have been
identified on the protein and on the mRNA level with a
lower abundance of PKC-z [14]. PKC-b has been found
inconsistently and preferentially in the developing heart
[14,15].
Whether cardiac hypertrophy potentially initiated by
activation of PKC may in turn modulate the differential
expression of PKC isozymes was the focus of the present
study. Of special interest were the temporal changes of
PKC isoform expression both at the protein and the mRNA
level in the isolated left and right ventricle relative to the
development of myocardial hypertrophy. Banding of the
ascending aorta was used as a well standardized experimental model for pressure-overload cardiac hypertrophy.
53
clips with a defined internal diameter of 0.711 mm
(Edward Weck, Research Triangle Park, NC, USA). Shamoperated animals underwent the same surgical procedure
without insertion of the clip. The perioperative mortality
was ¯10%. All rats were housed and fed according to the
¨
guidelines for animal care of the State of Baden-Wurttemberg. Hemodynamic and biochemical data were obtained 1,
7 or 30 days after the operation. To determine the left and
right ventricular enddiastolic pressure (LVEDP, RVEDP)
the chest was opened and a 0.6 mm stainless steal cannula
connected to a pressure transducer was inserted through
the free ventricular wall to record the pressure value on a
polygraph (PRC 21 and MVO-0600, FMI, Seeheim / Oberbeerbach, Germany; WeKegraph 350R, WKK, Kaltbrunn,
Switzerland). Thereafter, the heart and the lung were
quickly removed and the organ wet weights (whole heart,
the left and right ventricle and the lung) were determined
rapidly before freezing in liquid N 2 . The tissue was stored
at 280 8C until further use.
2.3. Determination of serum norepinephrine
One milliliter of blood was obtained at the time of
operation from the V. cava inferior and immediately
centrifuged for 5 min at 3003g. Perchloric acid (0.6 N)
was added to the plasma (1:1, v / v) and precipitated
proteins were sedimented by centrifugation (10003g, 5
min, 4 8C). The norepinephrine concentration in the
supernatant was determined using radio-enzyme assay
¨
according to the method of Da Prada and Zurcher
[16].
2.4. Preparation of the cytosol and particulate fractions
2. Methods
2.1. Materials
PKC-isoform-specific peptide antibodies were obtained
from St. Cruz (Heidelberg, Germany) and the enhanced
chemiluminescence (ECL) Western blotting detection reagents from Amersham International (Braunschweig, Germany). Reverse Transcriptase and Taq-DNA-Polymerase
were from Gibco (Germany), RNAse-Inhibitor, hexanucleotide mix and restriction enzymes from Boehringer
(Mannheim, Germany) and the oligonucleotides for PCR
and dNTPs from Pharmacia (Freiburg, Germany). All
other reagents were of analytical grade obtained from
Sigma (Munich, Germany). Male Wistar rats (¯90 g) were
purchased from Thomae (Biberach, Germany).
2.2. Aortic banding
Anaesthesia of the male Wistar rats was performed with
ketamine (100 mg / kg, i.p.). After left hemithoracotomy,
the ascending aorta was banded using tantalum hemostatic
Frozen pulverized tissue was dissolved in 2 ml 50 mM
Tris–HCl (pH 7.4), 100 mM sucrose, 10 mM b-mercaptoethanol, 1 mM phenylmethan-sulfoacidflouride, 1 mM
benzamidine, 1 mM EDTA, and 1 mM EGTA (buffer A).
The tissue was homogenized at 10,000 rpm three times for
6 s (Polytron LS-10-35, Kinematica, Luzern, Switzerland)
and centrifuged (3603g, 4 8C, 10 min). The resulting
supernatant was filtered through three layers of cheese
cloth and centrifuged (100,0003g, 4 8C, 60 min) to
separate the soluble fraction from the particulate fraction.
The pellet corresponding to the membrane fraction was
solubilized in buffer A containing Triton X-100 at a final
concentration of 0.1% by stirring on ice for 45 min.
Insoluble membrane particles were sedimented by centrifugation (100,0003g, 60 min, 4 8C). Triton X-100 was
added to the cytosol fraction as concentrated stock to give
a final concentration of 0.1%. Protein concentrations were
determined according to the method of Bradford using
bovine serum albumin as standard [17]. All samples were
frozen in aliquots at a final protein concentration of ¯5–12
mg / ml and stored at 280 8C.
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M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
2.5. Determination of PKC activity
PKC activity was determined in the cytosol and the
particulate fraction using the phosphorylation of histoneIII-S as a PKC substrate according to the method of Takai
et al. [18]. Basal PKC activity was measured in the
presence of 10 mM EDTA and 10 mM EGTA, and the
maximally stimulated PKC activity in the presence of 1.25
mM CaCl 2 , 100 mg / ml phosphatidylserine (PS) and 20
mg / ml 1,2-sn-diacylglycerol. The reaction (5 min, 30 8C)
was initiated by the addition of cytosol (¯5 mg protein) or
solubilized membranes (¯5 mg protein) and stopped on ice
with 20 ml 25 mM ATP. Aliquots of 60 ml were spotted on
Whatman P81 paper, washed (¯20 ml H 2 O / paper) and
¨
counted in a b-counter (Berthold BF 8000, Munchen,
Germany) using Cerenkov decay.
[19]. To verify the specificity of the amplification products,
restriction digests (1 h, 37 8C) were carried out with
PKC-isozyme-specific restriction enzymes, resulting in a
specific restriction pattern for each PKC isoform. Desmin
was used as internal standard. To allow absolute quantification of specific PKC mRNA levels, synthetic external
RNA standards with increasing concentrations were analyzed in parallel in all experiments. For the synthesis of the
RNA standards, see Strasser et al. [19]. All PCR amplification products were separated on a 2% agarose gel containing 0.01% ethidium bromide and visualized by UV
radiation. The electrophorized DNA fragments were vacuum blotted on a NY-13 nitrocellulose membrane and the
blots hybridized with [g 32 P]-labeled internal oligonucleotides to further confirm the specificity of the amplified
PCR products and to allow quantification.
2.6. Immunoblot analysis
2.8. Data analysis
Western blot analysis was performed on the cytosol and
the particulate fraction of the rat left and right ventricle as
previously described by Strasser et al. [19] using PKCisozyme-selective polyclonal antibodies (St. Cruz). The
specificity of the individual immunolabeled bands was
confirmed by their complete displacement in the presence
of the immunogenic peptide. As positive control the
cytosol fraction of rat brain was analyzed in parallel. To
ensure that equal amounts of protein were loaded on the
SDS–PAGE, the nitrocellulose blots were stained with
Ponçeau S solution (Serva / Heidelberg) at a final concentration of 0.2% in 3% trichloracetacid for 2 min at
room temperature. After washing the blot for 10 min in 20
ml water the proteins on the blot were visible. The protein
content in all blots did not vary by more than 10%.
Statistical analysis was performed using analysis of
variance and the Student–Newman–Keels test for significance [22].
2.7. RNA preparation, reverse transcription and
polymerase chain reaction ( RT-PCR) and synthetic RNA
standard
RNA extraction from the left and right ventricle was
performed according to the guanidine isothiocyanate ethanol precipitation method of Chirgwin et al. [20]. The purity
of the RNA probes were determined by UV absorption at
260 and 280 nm with a 260 / 280 yield .1.7 for all
samples.
Total RNA was reverse transcribed into cDNA using a
modified protocol of the reverse transcription polymerase
chain reaction method (RT-PCR) as described by Ponzoni
[21] and modified by Strasser et al. [19]. Two concentrations of template RNA (15 and 30 ng / ml) were chosen
to establish for each sample that the amplification procedure was performed in the linear range. For the polymerase chain reaction, the sense primers (SP) and antisense primers (ASP), and for Southern blot hybridisation
of the individual internal oligonucleotides specific for each
PKC isoform, were selected according to Strasser et al.
3. Results
3.1. Cardiac hypertrophy and hemodynamic parameters
Experimental banding of the ascending aorta in male
Wistar rats resulted in a rapid development of left ventricular hypertrophy with a significant increase in the left
ventricular index (LV weight / body weight) compared to
sham-operated animals (Table 1). A significant enhancement of the LV index was observed as early as 1 day after
aortic banding, with a further increase after 7 days and 30
days. Right ventricular hypertrophy, as determined by the
right ventricular index, occurred in the aortic stenosis
group 30 days after the operation. To verify if aortic
banding-induced cardiac hypertrophy is associated with
temporal changes of hemodynamic parameters and sympathetic stimulation, the left and right ventricular enddiastolic
pressures (LVEDP, RVEDP), the lung index (lung wet
weight / body weight; see Table 1) and plasma norepinephrine concentrations were determined. One and 30 days
after aortic banding, the LVEDP was significantly increased in the hypertrophied left ventricles to 135 and
236%, respectively. The enhanced LVEDP in the aortic
stenosis group 1 day after the operation is most likely due
to the rapid afterload elevation, resulting from acute
banding of the ascending aorta, followed by an early lung
edema as determined by an increased lung index. In
contrast, the increased LVEDP after long-term aortic
banding (30 days) reflects chronic left ventricular deterioration and is associated with a markedly enhanced lung
index, indicating chronic pulmonary congestion. Due to the
smaller size of the right ventricular cavum, reliable values
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
55
Table 1
Ventricular hypertrophy and hemodynamic parameters in pressure-overload cardiac hypertrophy
Parameter
LV index (g / kg)
RV index (g / kg)
LVEDP (mmHg)
Lung index (g / kg)
1 day
7 days
30 days
Sham
(n57)
AB
(n57)
Sham
(n59)
AB
(n59)
Sham
(n59)
AB
(n59)
2.8160.1
0.660.1
6.560.8
6.060.3
3.2860.08*
0.6260.07
8.861.1*
8.260.6*
2.3660.09
0.4960.08
6.460.9
5.960.9
3.7860.19**
0.6060.11
7.360.7
6.260.7
2.1960.14
0.4460.05
4.760.6
4.160.5
3.6960.21**
0.7360.1**
11.161.2**
7.961.0*
The left and right ventricular indices (left and right ventricular weight / body weight), the left ventricular enddiastolic pressures (LVEDPs) and the lung
index are shown for the sham-operated group (Sham) and the aortic banding group (AB) 1, 7 and 30 days after the operation. Values are mean6S.E.M.
*P,0.05, **P,0.01.
of the RVEDP were determined in both groups only 30
days after intervention, demonstrating a significant increase of the RVEDP in the chronic aortic stenosis group
(5.660.8 mmHg, n59) vs. the control animals (2.860.3
mmHg, n59, P#0.05). However, overt signs of heart
failure such as pleura effusion or abdominal ascitis were
not observed.
The plasma norepinephrine concentration was determined as a parameter for the activation of sympathetic
adrenergic activity. Thirty days after aortic banding the
plasma norepinephrine levels were significantly increased
to 184% (0.5960.09 vs. 0.3260.03 nmol / l, n59, P#
0.05) compared to controls, whereas at earlier time points
in both groups no significant changes were noted.
3.2. PKC activity in the subcellular fractions
The specific cardiac PKC activity in the cytosol fraction
of the left ventricle determined from sham-operated animals decreased from 188642 pmol /(mg protein?min) on
day 1 after the operation with further adolescence of the
young rat to 102624 pmol /(mg protein?min) on day 30
after the operation (Fig. 1, left panel). This developmental
decrease of the specific PKC activity was significant in the
control and the aortic banding group. After aortic banding
the specific PKC activity in the cytosol fraction was not
markedly altered compared to age-matched controls. Similarly, no significant differences of specific PKC activity
were observed in the particulate fractions after banding of
the ascending aorta [1 day after operation, 144635 vs.
166652 pmol /(mg protein?min), n.s.; 30 days after operation, 168648 vs. 145638 pmol /(mg protein?min), n.s.].
These data show that, in the hypertrophied myocardium,
the relative intracellular distribution of PKC activity and
specific PKC activity remained unaltered after aortic
banding. It should be mentioned that the specific PKC
activity reflects the maximally stimulated PKC activity
within these fractions and does not differentiate between
the distinct stimulation patterns of the individual PKC
isozymes or basal activity in vivo.
In contrast, the total cardiac PKC activity calculated for
the whole heart tended to increase 1 day after aortic
Fig. 1. Specific PKC activity (left panel) and total PKC activity (right panel) of the cytosol fraction of the left ventricule were determined by
phosphorylation of histone-III-S as substrate for PKC after sham operation and aortic banding. Values are mean6S.E.M. *P,0.05.
56
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
banding, without reaching significance (Fig. 1, right
panel). Upon further development of cardiac hypertrophy
the cardiac PKC activity of the whole heart was significantly increased 7 and 30 days after aortic banding by
57 and 88%, respectively. With the specific PKC activity
remaining constant, these data indicate that the increased
total enzyme activity is due to the coordinate increase of
protein in the course of hypertrophic development.
3.3. Expression of cardiac PKC isoforms after banding
of the ascending aorta
3.3.1. Left ventricle
The expression of the cardiac PKC isoforms in the
cytosol and membrane fraction of the left ventricular
myocardium was determined during the development of
cardiac hypertrophy after aortic banding using Western blot
analysis with PKC isoform subtype-specific peptide antibodies. All PKC isoforms were labeled as dominant bands
of the expected sizes, PKC-a at 80 kD, PKC-d as a doublet
at 78 and 80 kD, PKC-´ at 94 kD and PKC-z at 84 kD.
Fig. 2 shows a representative Western blot of the left
ventricular cytosol fraction for PKC-a, PKC-d and PKC-´
at the respective intervals after the operation. The density
of the bands labeled for PKC-a and PKC-d was increased
7 and 30 days after aortic constriction compared to agematched controls, whereas the density of the band for
PKC-´ in the cytosol fraction was not changed in both
groups at all indicated time points.
Taking into consideration the slight variations of the
protein loading (,10%) as detected by Ponçeau S staining
of the nitrocellulose blots, a quantitative analysis of the
complete set of experiments is shown in Table 2 (upper
panel). Aortic banding for 1 day did not alter the protein
expression of PKC-a, PKC-d and PKC-´ in the cytosol
fraction of the hypertrophied left ventricle compared to
controls, whereas after 7 and 30 days a significant increase
of PKC-a (212 and 193% vs. control) and PKC-d (267 and
Fig. 2. Representative Western blot analysis of the PKC isoforms PKC-a, PKC-d and PKC-´ in the left ventricle after sham operation (sham) or aortic
banding (AB). An increased protein amount for PKC-a and PKC-d, but not for PKC-´, was detected 7 and 30 days after banding of the ascending aorta.
The specificity of the immunoreactive proteins was established in the presence of competing immunogenic peptide (1p) and a positive control (brain). The
positions of the molecular mass standards (in kD) are indicated on the right.
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
57
Table 2
Quantification of PKC isoform expression in aortic banding-induced hypertrophy
1 day
7 days
30 days
Cytosol
PKC-a
PKC-d
PKC-´
94615% (n57)
122627% (n57)
108610% (n57)
212620% (n58)*
267618% (n58)**
132614% (n58)
193614% (n58)*
289612% (n59)**
115615% (n59)
Membrane
PKC-a
PKC-d
PKC-´
137612% (n57)
120615% (n57)
89618% (n57)
141612% (n58)
177618% (n58)*
102611% (n58)
12368% (n58)
249619% (n59)**
110619% (n59)
The protein content of each PKC isozyme was determined in the left ventricular cytosol fraction (upper panel) and the membrane fraction (lower panel)
of the left ventricle after sham operation or aortic banding. The numbers represent the relative percentage of the PKC isoform amount in the aortic banding
group compared to controls; ‘‘n’’ indicates the number of experiments performed for each PKC isoform. The data are expressed as mean6S.E.M.
*P,0.05, **P,0.01.
289% vs. control) was observed. In contrast, the expression of PKC-´ remained unaltered in both groups up to 30
days after intervention.
In the membrane fraction of the hypertrophied left
ventricle a different protein regulation of PKC isozyme
expression was observed (Table 2, lower panel). The
amount of immunodetectable protein of PKC-a and PKC-´
in the membrane fraction was not significantly changed up
to 30 days after aortic banding compared to controls.
However, similar to regulation in the cytosol fraction,
PKC-d was significantly increased 7 and 30 days after
banding of the ascending aorta to 177618 and 249619%,
respectively. For the atypical PKC isoform PKC-z, significant changes in the cytosol and particulate fractions were
not detectable in either experimental group (data not
shown).
3.3.2. Right ventricle
Immunoblot analysis of the PKC isoforms in the right
ventricle revealed expression of PKC-a, PKC-d and PKC´ in the sham-operated and aortic banding group, as
demonstrated in Fig. 3. Banding of the ascending aorta for
30 days resulted in right ventricular hypertrophy (Table 1).
This was associated with an increased density of PKC-a
and PKC-d in the cytosol fraction, whereas PKC-´ was
unchanged in either group at the respective time points
(Fig. 3). Statistical evaluation for the dominant right
ventricular PKC isoforms demonstrates a significant increased protein expression for PKC-a (156621%) and
PKC-d (179628%) in the cytosol fraction 30 days after
aortic banding vs. age-matched controls (Table 3, upper
panel). However, in the membrane fraction of the right
ventricle, an enhanced protein amount was detected only
for PKC-d (168629%), whereas PKC-a tended to increase
7 and 30 days after aortic banding without reaching
significance (Table 3, lower panel). PKC-´ was not
significantly changed in either group. Similar to the protein
expression pattern in the hypertrophied left ventricle, these
data suggest an important role of PKC-a and PKC-d in the
development of right ventricular hypertrophy following left
ventricular failure due to long-term aortic banding.
3.4. Quantitative mRNA expression of cardiac PKC
isoforms after banding of the ascending aorta
To investigate if the increased protein expression of
PKC-d and / or PKC-a at the protein level may be due to
an upregulation of the specific mRNAs, the mRNA levels
for the dominant cardiac PKC isoforms PKC-a, PKC-d,
PKC-´ and PKC-z were determined in the left and right
ventricle of sham-operated controls and after aortic banding up to 30 days. Absolute quantification of the PKCisoform-specific mRNA levels was determined by a newly
established RT-PCR method using external and internal
RNA standards [19].
3.4.1. Left ventricle
A representative agarose gel of the RT-PCR analyses of
PKC-d and PKC-´ from the left ventricle of sham-operated
controls and after aortic banding is shown in Fig. 4 (left
panel). The density of the PKC-d RT-PCR product is
increased 1, 7 and 30 days after aortic banding compared
to age-matched controls, suggesting an upregulation of
PKC-d mRNA in pressure-overload-induced left ventricular hypertrophy. In contrast, no significant differences of
the PKC-´ mRNA levels were detected between the
experimental groups.
The quantitative analysis of the whole set of experiments is shown in Fig. 5 for all four PKC isozymes
analyzed in the left ventricle. As early as 1 day after aortic
banding the absolute mRNA level for PKC-a tended to
increase without reaching significance at that early point in
time (Fig. 5A), whereas after 7 and 30 days the absolute
amount of PKC-a mRNA was significantly increased by
64 and 77% of controls, respectively. In addition, aortic
banding for 1 day promoted a significantly increased
expression of PKC-d mRNA by 57%, which was even
more pronounced after 7 (193%) and 30 days (202%, Fig.
5B). However, the mRNA levels for PKC-´ and PKC-z
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
58
Fig. 3. Representative immunoblot analysis of PKC-a, PKC-d and PKC-´ in the right ventricle after sham operation (sham) or banding of the ascending
aorta (AB). Equal protein aliquots from the cytosol fraction were analyzed by SDS–PAGE. The specificity of the immunoreactive bands was determined
using the competing peptide (1p). The molecular mass standards (in kD) are indicated on the right.
remained unaltered in both groups (Fig. 5C and D). The
absolute levels of PKC-isoform-specific mRNA were in
the range 1200 ag / ng total RNA for PKC-´, 750 ag / ng
Table 3
Quantitative analysis of PKC isoform expression in the cytosol and
membrane fraction of the right ventricle after sham operation or aortic
banding
1 day
7 days
30 days
82616% (n57)
102619% (n57)
91612% (n57)
125638% (n58)
132641% (n58)
11368% (n58)
156621% (n58)*
179628% (n58)*
100614% (n57)
Membrane
PKC-a
89621% (n57)
PKC-d
9768% (n57)
PKC-´
111625% (n57)
136632% (n58)
127640% (n57)
92619% (n58)
121637% (n58)
168629% (n58)*
122610% (n57)
Cytosol
PKC-a
PKC-d
PKC-´
The protein amount of individual PKC isozymes in the aortic stenosis
group is expressed as a percentage of the respective density in the
sham-operated group; ‘‘n’’ indicates the number of experiments. The
results are expressed as mean6S.E.M. *P,0.05.
total RNA for PKC-d and 200 ag / ng total RNA for
PKC-a. PKC-z mRNA was in the range 60 ag / ng total
RNA, suggesting a relatively lower abundance of this PKC
isoform in rat myocardium. This corresponds to the finding
that, in Western blot analysis, the expression of PKC-z
protein was very low.
3.4.2. Right ventricle
The results from the mRNA analyses of the cardiac PKC
isoforms in the right ventricle after sham operation or
aortic banding are summarized in Table 4. In parallel to the
protein expression of the individual PKC isoforms, the
absolute mRNA levels of PKC-a and PKC-d were unchanged after 1 and 7 days but markedly increased after 30
days of aortic banding by 46 and 54% compared to
sham-operated controls. However, no significant changes
were observed for the amount of PKC-´ mRNA in either
group.
Direct comparison of the PKC-isoform-specific mRNA
levels in the right and left ventricle of the sham-operated
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
59
Fig. 4. Representative RT-PCR products for PKC-d and PKC-´ of the left ventricle after sham operation (sham) or aortic banding (AB). The mRNA level
of PKC-d was increased 1, 7 and 30 days after aortic banding vs. control (upper left panel), whereas for PKC-´ no relevant changes in the mRNA level
were detectable in either group (lower left panel). For quantification, PKC-isoform-specific RNA standards were analyzed in parallel (right panel).
Fig. 5. Subtype-specific regulation of mRNA for PKC-a, PKC-d, PKC-´ and PKC-z after the respective times of sham operation or aortic banding.
RT-PCR of total mRNA of the left ventricle and Southern blot hybridization were performed. For quantification, the autoradiograms were analyzed by laser
densitometry and in correlation with the RNA standards for each PKC isoform the amount of specific mRNA was calculated. The data are expressed as
mean6S.E.M. *P,0.05, **P,0.01.
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
60
Table 4
Quantification of the specific mRNA steady-state levels (ag / ng total RNA) for the dominant cardiac PKC isozymes in the right ventricle after sham
operation or aortic banding
PKC
isoform
1 day
7 days
30 days
Sham
(n56)
AB
(n56)
Sham
(n58)
AB
(n58)
Sham
(n58)
AB
(n58)
PKC-a
PKC-d
PKC-´
275643
559664
941671
225631
503634
1151695
228628
454643
1011635
260611
478641
902667
204611
384629
822652
298617*
591618*
739628
The absolute mRNA levels were determined using mRNA standards for each PKC isoform as demonstrated for the left ventricle. Data are expressed as
mean6S.E.M. *P,0.05.
controls revealed similar absolute values for PKC-a, PKCd and PKC-´ mRNA, which might be due to the closely
related transcriptional regulation processes in both myocardial tissues.
4. Discussion
Cardiac hypertrophy is a general physiological adaptation process to increased myocardial work. To investigate
if, during the development of cardiac hypertrophy, the
expression of PKC, an important intracellular growthpromoting enzyme, may be modulated we have characterized the differential expression of cardiac PKC isozymes in
pressure-overload-induced left ventricular hypertrophy due
to aortic banding.
Early during the development of left ventricular hypertrophy, PKC-d and PKC-a are increased both at the
protein and the mRNA level, whereas PKC-´ and PKC-z
remained unaltered. The atypical PKC isoform PKC-z was
expressed at a very low level, which is in good agreement
with previously published data [14,23]. The selective
regulation of PKC isoforms at the mRNA level validates
the regulation at the protein level. The increased mRNA
amount of PKC-d 1 day after aortic banding indicates that
the increased expression of PKC-d is initiated at a very
early stage of LV hypertrophy. In contrast, 1 day after
aortic banding the mRNA level of PKC-a tended to
increase, but was not significantly different from the agematched controls. These data indicate that PKC-d might
play a role in the initiation of hypertrophy, whereas
isoforms PKC-d and PKC-a may support the maintainance
of pressure-induced cardiac hypertrophy. Similar results
were found for right ventricular hypertrophy resulting from
chronically elevated left ventricular enddiastolic pressure
due to long-term banding of the ascending aorta, demonstrating selective upregulation of PKC-a and PKC-d.
Furthermore, enhanced protein expression of PKC-a and
PKC-d could also be detected in volume-overload cardiac
hypertrophy due to experimental aorto-caval shunt as well
as chronic treatment with isoproterenol (unpublished data).
Possibly, due to the very low abundance of PKC-b in
the adult rat heart, this isozyme could not be detected by
Western blot analysis (data not shown). Additionally, no
PKC-b was identified after the development of pressureoverload left ventricular hypertrophy. However, there is
considerable controversy about the expression of PKC-b in
adult rat heart [14,19,23–27]. A study by Gu and Bishop in
Sprague–Dawley rats demonstrated the presence of PKC´, PKC-d and PKC-b in the left ventricle of sham-operated
controls and increased expression of PKC-b in the aortic
stenosis group 2 weeks after intervention [27]. However,
the immunolabeled bands of PKC-b did not comigrate
with the brain standard. Moreover, Gu and Bishop used a
different rat strain at a single time point after aortic
constriction, which may further contribute to their different
results. A recent evaluation by Roman et al. showed, in
PKC-b knockout mice, that PKC-b expression is not
necessary for the development of cardiac hypertrophy
induced by different and independent hypertrophic stimuli
such as phenylepinephrine infusion or aortic banding [28].
Differential roles for individual PKC isoforms in the
pathogenesis of cardiac hypertrophy and congestive heart
failure have been reported by several authors. Cardiacspecific overexpression of a constitutively active mutant of
PKC-´ in trangenic mice lead to a concentric hypertrophy
with normal in vivo function of the left ventricle [29]. A
recent study by Mende et al. supports our data showing
that overexpression of a constitutively active mutant of the
Gaq subunit in the heart of transgenic mice is sufficient to
induce cardiac hypertrophy, which is associated with an
upregulation of PKC-a and PKC-d isozymes without an
enhancement of PKC-´ [30]. Furthermore, overexpression
of a Gaq inhibitor peptide prevented cardiac hypertrophy
through pressure-overload stimuli [31].
The increased protein expression of PKC-d after aortic
banding was shown both in the cytosol and the particulate
fraction, whereas PKC-a was enhanced only in the cytosol
fraction. Previous studies have demonstrated that acute
activation and stimulation of PKC is generally associated
with intracellular translocation of the enzyme from the
cytosol to the plasma membrane, indicating its rapid
activation [32]. However, since translocation has been
shown to be a transient phenomenon [33], constitutive
activation of PKC in cardiac hypertrophy may not be
reflected by chronic translocation. The data of the present
study might be explained by a concomittant dissociation of
the enzyme from the membrane back to the cytosol
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
without significant degradation, suggesting that the cytosol
not only represents a ‘‘resting’’ compartment for inactive
PKC, but may also contain compartments where PKC
isozymes may be active [34].
However, the signaling pathways in cardiac myocytes
leading to cardiac hypertrophy due to aortic banding are
not well defined. It has previously been reported that
angiotensin II (ANG II) may have a direct growth effect on
ventricular myocytes and may play a role in cardiac
hypertrophy due to pressure-overload [35]. Treatment with
the angiotensin-converting enzyme inhibitor lisinopril or
enalapril resulted in almost complete prevention or reversal
of pressure-overload cardiac hypertrophy [36,37]. It was
demonstrated by Sadoshimo that stretching of cultured
cardiac myocytes releases ANG II into the medium, which
can then activate specific ANG II receptors [38]. Activation of ANG II receptors results in a biphasic activation of
PKC, reflected by an early and rapid translocation of PKC
with an initial increase of the membrane-bound and a
decrease in the cytosolic PKC activity. Long-term stimulation of cardiac myocytes with ANG II leads to a concomitant upregulation of cytosolic PKC [39]. It is conceivable
that, in the present study, a similar biphasic activation
process may have initiated the augmented expression of
PKC-a and PKC-d. However, it has not been demonstrated
whether, in the present model, ANG II is responsible for
this regulation process. Using ANG II type 1 (AT1)
receptor knockout mice as an experimental animal model,
Harada questioned a significant etiologic component of the
renin–angiotensin system in the development of cardiac
hypertrophy due to pressure-overload [40]. Banding of the
ascending aorta results in the same degree of myocardial
hypertrophy in AT1-receptor-deficient mice as observed in
wild type. These data provide evidence that the hypertrophic response in the model of aortic banding is mediated
through signaling transduction pathways other than AT1
receptor stimulation. Other signaling molecules which
might represent a linkage between pressure-overload and
the cellular hypertrophic response include endothelin-1,
diverse plasma membrane ion-channels [41] and calcineurin [42].
Calcineurin, a calcium-regulated serine / threonine-specific phosphatase, induces the activation of nuclear transcription factors via dephosphorylation of NFAT (nuclear
factor of activated T cells) [42]. Several authors have
identified an important role of calcineurin in the development of cardiac hypertrophy using overexpression models
and pharmacological inhibition of the enzyme [43–45]. In
contrast to these studies, other authors have concluded that
calcineurin inhibitors such as cyclosporine have no effect
in blocking pressure-overload cardiac hypertrophy in rodents [46,47]. These conflicting results might be explained
by (a) differences in experimental protocols, (b) a reflection of the truly distinct roles of calcineurin in the
pathogenesis of diverse hypertrophic models or (c) the
different compensated or decompensated state of cardiac
hypertrophy.
61
However, calcineurin probably regulates the hypertrophic response in coordination with other intracellular
transduction signals, including the c-Jun N-terminal kinase,
MAPK and PKC-a in cardiac myocytes [48]. Furthermore,
calcineurin is known to be involved in the regulation of
intracellular calcium dynamics, partially by modulating the
function of the sarcoplasmic reticulum ATPase (SERCA),
which is a central protein for the reuptake of Ca 21 in
cardiomyocytes. Decreased SERCA activity is associated
with alterations in Ca 21 cycling and contractile dysfunction [49]. Munch et al. recently demonstrated, in human
congestive heart failure, that the enhanced level of calcineurin contributes to a decrease in SERCA 2a activity
and impaired Ca 21 handling in dilated cardiomyopathy
[50]. The depressed SERCA activity leads to decreased
Ca 21 uptake and might play an important role in the
transition from cardiac hypertrophy to heart failure. The
regulation of SERCA by calcineurin is in accordance with
data from Xu et al. [51], but is in contrast to other data
[52].
In addition to these signaling pathways, several authors
have reported an independent role of the cardiac b-adrenergic system in the development of myocardial hypertrophy and heart failure [42,53,54]. A recent study by
Borst et al. demonstrated that the contractile function of
isolated rat cardiomyocytes in pressure-overload cardiac
hypertrophy is diminished due to desensitisation of the
stimulatory side of the adenylyl-cyclase signal transduction
system [53]. Additionally, Sumida found an increased
Ca 21 transient and a reduced myofilament responsiveness
to Ca 21 in hypertrophied cardiac myocytes [54]. At this
point it is of special interest that PKC-a and PKC-d are
known to modulate the Ca 21 responsiveness of cardiac
myofilaments by phosphorylation of specific serine /
threonine sites of troponin I (TnI), an important protein of
the contractile apparatus [55]. Furthermore, the phosphorylation sites of TnI mediated by PKC-d (ser23 / ser24)
are identical to the phosphorylation site of PKA [55],
suggesting that both signal pathways regulate contractile
function at the level of troponin I.
It should be mentioned that the hypertrophic response
due to an increased mechanical load of the adult rat heart
differs from postnatal physiological growth and seems to
be more a pathological process than well compensated
accelerated growth [56]. Maturation from the fetal to the
adult left ventricle is accompanied by physiological
growth, whereas experimental aortic stenosis-induced cardiac hypertrophy of the adult rat heart results in concentric
and / or excentric hypertrophy. These morphological
changes are also reflected at the molecular level and PKC
might have different roles in both forms of cell growth.
During postnatal development, decreases of PKC activity
and PKC isozyme expression in the neonatal rat heart have
been described by Clerk et al. [57]. In contrast, upregulation of selective PKC isozymes in pressure-overload
cardiac hypertrophy has been identified in the present
study and by others [27]. These data suggest that the
62
M.U. Braun et al. / Cardiovascular Research 56 (2002) 52–63
regulation of PKC or selective PKC isozymes varies in
physiological growth and pathophysiological hypertrophy.
Identification of the PKC isozymes which are upregulated during pressure-overload left ventricular hypertrophy
might provide the opportunity to delay or prevent the
hypertrophic process. Due to the detected transcription
regulation of PKC isozyme expression in aortic banding,
the authors favor a causal approach to the intervention of
the hypertrophic remodeling process by selectively and
completely blocking individual gene transcripts of specific
PKC isozymes rather than the use of selective PKC protein
inhibitors [58]. Further investigations are necessary to
evaluate whether or not this approach will provide new
therapeutic strategies.
As a limitation of the present study it should be
mentioned that differentiation of PKC isozyme expression
at the cellular level of myocardial tissue (cardiac myocytes
vs. non-myocytes) was not possible. However, since 60–
80% of the mass of cardiac tissue is represented by cardiac
myocytes, it appears unlikely that non-myocytes were a
major source of PKC. However, we cannot exclude a
contribution of PKC isozyme expression from the nonmyocyte population in this model of cardiac hypertrophy.
In summary, pressure-overload cardiac hypertrophy due
to banding of the ascending aorta in rats is accompanied by
a selective increase of PKC-d and PKC-a expression both
at the mRNA and at the protein level. In contrast, the
mRNA and protein levels of PKC-´ and PKC-z are not
significantly altered in hypertrophied left ventricles compared to age-matched controls. Identification of the differential expression of PKC isozymes in the right and left
ventricle during the development of pressure-overload
cardiac hypertrophy might give further insight into the
signal transduction pathways responsible for stretch-induced hypertrophic growth and might provide new therapeutic targets for PKC-isozyme-selective PKC inhibitors
and activators.
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, SFB 320. RHS was supported by the
Herrman and Lilly Schilling Foundation. The authors
would like to thank Ulrike Oehl and Annette Kempkes for
expert technical assistance.
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