Vol. 284, Issue 4, H1168-H1181, April 2003
Role of PKC in autocrine regulation of rat ventricular
K+ currents by angiotensin and endothelin
Yakhin
Shimoni and
Xiu-Fang
Liu
Cardiovascular Research Group, Department of Physiology and
Biophysics, University of Calgary, Calgary, Alberta,
Canada T2N 4N1
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ABSTRACT |
Transient and sustained
K+ currents were measured in isolated rat ventricular
myocytes obtained from control, steptozotocin-induced (Type 1)
diabetic, and hypothyroid rats. Both currents, attenuated by
the endocrine abnormalities, were significantly augmented by in vitro
incubation (>6 h) with the angiotensin-converting enzyme inhibitor
quinapril or the angiotensin II (ANG II) receptor blocker saralasin.
Western blots indicated a parallel increase in Kv4.2 and Kv1.2, channel
proteins that underlie the transient and (part of the) sustained
currents. Under diabetic and hypothyroid conditions, both currents were
also augmented by an endothelin receptor blocker (PD142893) or by an
endothelin-converting enzyme inhibitor. Kv4.2 density was also enhanced
by PD142893. Incubation (>5 h) with the PKC inhibitor
bis-indolylmaleimide augmented both currents, whereas the PKC activator
dioctanoyl-rac-glycerol (DiC8) prevented the augmentation of currents
by quinapril. DiC8 also prevented the augmentation of Kv4.2 density by
quinapril. Specific peptides that activate PKC translocation indicated
that PKC-
and not PKC-
is involved in ANG II action on these
currents. In control myocytes, quinapril and PD142893 augmented the
sustained late current but had no effect on peak current. It is
concluded that an autocrine release of angiotensin and endothelin in
diabetic and hypothyroid conditions attenuates K+ currents
by suppressing the synthesis of some K+ channel proteins,
with the effects mediated at least partially by PKC-.
diabetes; protein kinase C
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INTRODUCTION |
WE HAVE RECENTLY
ESTABLISHED that an autocrine release of angiotensin II (ANG II)
plays a central role in attenuating a transient and a sustained
K+ current in single myocytes isolated from (Type 1)
diabetic rats (45). The in vitro blockade of ANG II action
or formation (for >5 h) augments both currents. This effect is blocked
by cycloheximide, suggesting that new protein synthesis mediates
current augmentation after removal of ANG II-related current
inhibition. This protein synthesis may be of the pore-forming
-subunits of the channels, but not necessarily so. There is
currently an increasing awareness of the complexity of channel
function, with key roles established for 
-subunits as well as
various accessory and chaperone proteins. These can interact with the
pore-forming subunit of the channel and modulate its surface expression
(3, 27, 40, 50). Thus changes in current magnitude may
reflect alterations in these accessory proteins as well as changes in
channel processing or in posttranslation modification (e.g., 38). It is
therefore essential to directly establish whether an augmentation in
the -subunit of the channel protein can in fact be identified after
interference with ANG II action.
Several additional issues relating to the autocrine/paracrine control
of ion channels arise from the preceding work. ANG II has multiple and
diverse cellular actions (11). A key modulator of cellular
function, protein kinase C (PKC), is a major target of ANG II
(11). PKC is activated in diabetes, with several isoforms affected (22, 29). The -isoform of PKC is of particular
interest, because it is involved in several cardiac pathologies and
plays a key role in cardioprotection (10). Furthermore,
PKC- also interacts with K+ channels (33,
44). Diabetes leads to a change in the subcellular distribution
of PKC-, so that a greater proportion is found in membrane fractions
(29). This is prevented by ANG II receptor blockade
(29). The acute effects of PKC- activation on
K+ currents are altered in diabetes as well
(44). Alterations in K+ currents can lead to
severe cardiac arrhythmias (19, 27, 48). Thus it is of
importance to establish some of the mediating mechanisms, such as
whether the suppression of the two K+ currents by ANG II is
associated with PKC- activation and translocation.
Other pathological conditions that are associated with PKC-
activation and subcellular translocation also result in attenuated K+ currents. These include hypoxia and ischemia
(16) as well as cardiac hypertrophy (17). The
same transient and sustained currents are attenuated in ventricular
myocytes from hypothyroid rats (46), in which PKC- is
also activated (42). It was thus of interest to establish
whether ANG II is also a mediator of K+ current suppression
under conditions other than diabetes. Finally, there have been reports
indicating that after stress there is enhanced production and/or
release of other autocrine/paracrine agents in the heart. For example,
endothelin (ET)-1 is released (12), possibly from cardiac
myocytes as well as from other cell types (41).
The aims of this study were therefore to extend earlier work and
establish the following: 1) Does restoration of current
magnitudes after ANG II blockade involve the synthesis of new channels?
2) Does autocrine/paracrine regulation of K+
currents occur in other pathologies such as under hypothyroid conditions? 3) Do other agents such as ET-1 play a role in
the chronic suppression of K+ currents? 4) Is
PKC- involved in the chronic suppression of K+ currents
and, more specifically, does the restoration of current magnitudes
involve a return of PKC distribution to the normal (prediabetic,
euthyroid) state?
The present work suggests that the answer to all of these questions is affirmative.
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METHODS |
All of the experiments were done according to guidelines
established and approved by the Animal Care Committee of the University of Calgary. Rats were anesthetized by methoxyflurane or CO2
inhalation and killed by cervical dislocation. Hearts were removed, and
the aortas were cannulated for coronary perfusion on a Langendorff apparatus.
Animals.
Ventricular cells or tissue were obtained from three groups of adult
Sprague-Dawley male rats (200-300 g): untreated control rats, rats
made diabetic with a single intravenous injection of streptozotocin
(100 mg/kg) 8-14 days before the experiments, and rats made
hypothyroid by thyroidectomy 4-5 wk before the experiments.
Cell isolation.
Current recordings were done using isolated cells from the free wall of
the right ventricle, obtained by enzymatic dispersion using collagenase
(Yakult; Tokyo, Japan) and protease (Sigma type XIV). Details can be
found in an earlier publication (44).
Current recordings.
These were done using the whole cell suction electrode method.
Low-resistance electrodes (2-4 M
) were used to minimize series resistance, which was also compensated for electronically. Pipette solutions contained (in mM) 120 K-aspartate, 30 KCl, 4 Na2ATP, 10 EGTA, 1 CaCl2, 1 MgCl2,
and 10 HEPES (pH brought to7.2 with KOH). The perfusing solution
contained (in mM) 150 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 5.5 glucose, 5 HEPES, and 0.3 CdCl2 to
block L-type Ca2+ current. Two currents were recorded: the
calcium-independent transient outward current (34) and a
sustained delayed rectifier current. The transient current was measured
as the peak outward current (Ipeak) and not as
the difference between the peak and steady-state currents, because the
steady-state current was also subject to differential changes in these
experiments. The sustained current activates considerably slower than
Ipeak (4) and was measured at the
end of 500-ms pulses, at which time Ipeak is
completely inactivated. The late current is referred to as
Ilate. Recent analysis by Himmel et al.
(21) has shown that a mixture of four currents is present
in rat ventricular myocytes, and the effects of the
angiotensin-converting enzyme (ACE) inhibitor quinapril were analyzed
in detail using their methods (see below) to establish which components
are affected.
Data analysis.
Currents were recorded at 20-22°C (digitized at 2 kHz using a
DT2821 analog-to-digital board) and stored for subsequent analysis. Cell capacitance was measured for each cell by integrating current traces (digitized at 10 kHz) obtained in response to 5-mV steps from a
holding potential of 
80 mV. Dividing current magnitudes by
capacitance gave current densities.
To account for some variation in the diabetic state between rats,
currents were measured in untreated myocytes on each experimental day
as well as in myocytes from treated groups (giving slightly different
baselines for each protocol). The current densities in cells from each
experimental group were averaged and compared. Measurements were made
in all cells at +50 mV, at which the fast sodium current is negligible,
and at 110 mV. In many cells, full current-voltage relationships were
also obtained.
Earlier work by Himmel et al. (21) provided a
comprehensive analysis of the different current components by analyzing
the inactivation characteristics of total outward currents in rat ventricular cells. They showed that a protocol consisting of 2,000-ms prepulses to potentials ranging from 140 to +20 mV (in 10-mV steps
except for the range of 60 and 20 mV, in which 5-mV steps were
used), followed by test pulses to +60 mV, allowed the measurement of
steady-state inactivation of currents that could best be fit by two
Boltzmann functions. The normalized currents [current
(I)/maximal current (Imax)] were fit
to the equation
where a and b are the relative
contributions of the two functions and r is a residual component.
The inactivation process was analyzed separately for
Ipeak and Ilate at the
end of the test pulse [see Himmel et al. (21)]. This
analysis revealed the presence of four current components. The
parameters a and b (for
Ipeak) obtained by this analysis corresponded to
a delayed rectifier and a transient current, respectively, whereas
r reflected a noninactivating current.
Vm is membrane potential,
V0.5 is the half-inactivation potential, and
k is the slope factor (separate for each function).
Reagents.
Saralasin was obtained from Calbiochem, and PD142893 and the
ET-converting enzyme (ECE) inhibitor (fragment 16-38 of Big ET-1) were from Sigma. Quinapril was a gift from Dr. A. Gillis (Foothills Hospital, Calgary, Alberta, Canada). The PKC-related peptides were
obtained from Dr. D. Mochly-Rosen, Stanford University.
Western blotting.
The channel proteins encoded by Kv4.2 and Kv4.3 underlie the transient
outward current in rat ventricular cells (14, 34). The
sustained current has several components (21) and is the complex expression of several channel proteins (34). In
our experiments, antibodies directed against Kv4.2, Kv1.2 (Alomone), and Kv4.3 (Alomone and an antibody kindly provided by Dr. J. Nerbonne) were used. We were able to obtain consistent and reliable
data using the Kv4.2 and Kv1.2 antibodies. However, both antibodies directed against Kv4.3 gave bands of poorer quality, and the results could not be used. However, the results with the Kv4.2 and Kv1.2 antibodies were sufficient to establish that current changes are paralleled by changes in protein density (see below).
The membrane fractions of ventricular tissue (rather than cells) were
used to obtain sufficient protein (see DISCUSSION). Because
many of the effects observed on current magnitudes were only seen after
6 h, the following procedure was followed: aortas were cannulated,
and the hearts were perfused using a Langendorff apparatus (at 37°C,
70 cmH2O pressure) for at least 7 h in the absence or
presence of different compounds, as indicated below. After 7-9 h,
during which the hearts maintained rhythmic spontaneous beating, both
ventricles were dissected and frozen rapidly for subsequent analysis.
The ventricles were later thawed and (~0.4 g tissue) homogenized in
Dounce [on ice in a 10 mM Tris buffer containing 0.5 M sucrose and a
protease inhibitor tablet (Roche)]. The homogenate was centrifuged at
4°C for 15 min at 1,200 rpm to remove debris and nuclei. The
supernatant was removed and spun at 100,000 g for 1 h.
The membrane pellet was resuspended in Tris buffer containing 1%
Triton X-100. Trituration and vigorous vortexing were used to resuspend
the pellet until large particles were no longer visible. Protein
concentration in the membrane fraction was determined (using duplicates
for each sample) by the bicinchoninic acid assay (Pierce). Membrane
proteins (including positive and negative controls, see below) were
separated by 10% acrylamide SDS-PAGE and transferred to 0.45 µM
nitrocellulose membranes before being blocked with 5% nonfat dried
milk in a Tris-buffered saline (TBS) solution. Membranes were labeled
with rabbit polyclonal anti-Kv4.2 or Kv1.2 (diluted 1:400), washed with
TBS solution, and probed with 1:4,000 horseradish peroxidase-conjugated
goat anti-rabbit IgG. Detection was achieved with a chemiluminescent
substrate (Pierce) using Biomax film.
Quantification of Kv4.2 and Kv1.2.
SDS-PAGE gels used for transfer to nitrocellulose and Western blotting
were run simultaneously with gels loaded with 100 µg protein and
stained with Coomassie brilliant blue R-250 (Bio-Rad). Densitometry was
performed on the immunoreactive bands as well as on an arbitrary band
on the Coomassie blue protein gels to quantitatively normalize to
protein loading levels in all lanes. A Sharp JX-330 Scanner and Image
Master 1D Software (Pharmacia Biotech) were used. Each gel had two to
three samples from each experimental group, which allowed for
variations in optical density (OD) due to variations in film
development. Channel protein in the different groups (in OD units) was
normalized to the OD of the Coomassie blue band or to protein loading
levels (100 µg protein) and averaged for the different groups. Both
methods of normalization gave similar results.
To verify the identification of the correct bands on the gel
corresponding to Kv4.2, incubation of samples was also performed in the
presence of competing peptides. This eliminated the band for Kv4.2, but
other bands were also blocked (see below and Fig. 2). A more rigorous
control test was therefore used. Human embryonic kidney (HEK-293) cells
were transfected with cDNA for Kv4.2 using a pIRES-ht GFP-1a vector.
After protein expression, cells were homogenized, and human embryonic
kidney cell membrane extracts were assayed for Kv.4.2 alongside the
ventricular samples. The Kv4.2 bands from the HEK-293 cells were
located in parallel to the bands from ventricular tissue, confirming
the identity of the bands from the ventricular samples (Fig. 2). The
identity of the Kv1.2 band was confirmed using a competing peptide (see below).
Statistics.
An unpaired Student's t-test or (mostly) ANOVA with a
Student-Newman-Keuls multiple-comparison post hoc test were used to establish whether experimental groups differed (with P < 0.05 indicating a significant difference).
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RESULTS |
The first set of results, summarizing and extending earlier work,
demonstrates that the exposure of myocytes from diabetic rats to the
ACE inhibitor quinapril for 6 h or longer augments the peak
(transient) and late outward currents. Figure
1 shows sample current traces
(A) as well as the full current-voltage relationships
(B) for Ipeak (left) and
Ilate (right). Mean (±SE) current
densities are plotted against membrane potentials in the absence and
presence of quinapril (6- to 9-h incubation).
Ipeak was significantly augmented by quinapril
(P < 0.0005 for membrane potentials between 30 and
+50 mV). Ilate in this potential range was also
significantly enhanced (P < 0.005). There were no
effects of quinapril on Ilate in the negative
potential range (50 to 110 mV), in which the inward rectifying
background current is activated.
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Fig. 1.
Effects of an angiotensin-converting enzyme (ACE) inhibitor on
K+ currents in ventricular myocytes from diabetic rats.
A: sample current traces obtained in myocytes from a
diabetic rat in response to 500-ms depolarizing steps from 80 mV to
membrane potentials ranging from 0 to +50 mV. Left, traces
in an untreated cell; right, membrane potentials in a cell
exposed to 1 µM quinapril for 7 h. The line on the
left indicates the zero-current level. B:
current-voltage relationships showing the mean (±SE) densities for
peak currents (Ipeak; left) and late
currents (Ilate; right) in the
absence of quinapril (circles) or after 6-9 h in 1 µM quinapril
(squares). The differences for both currents were highly significant
between 30 and +50 mV (P < 0.0005 for
Ipeak; P < 0.005 for
Ilate).
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We subsequently attempted to analyze more precisely which current
components are modulated by quinapril. We repeated the analysis of
Himmel et al. (21) and analyzed the inactivation of the
total outward currents. Prepulses (2,000 ms) were given to potentials ranging from 140 to 20 mV in 10-mV steps (5-mV steps between 60
and 20 mV), followed by a 500-ms pulse to +50 mV. Current amplitudes
were normalized to maximal currents, and the data were fit to a sum of
two Boltzmann functions with the residual component r. The
analysis was done separately for Ipeak and
Ilate at the end of the test pulses. The
different parameters (see METHODS for the equation) were
compared in cells from diabetic rats in the absence (n = 25) or presence (n = 21) of quinapril (6-9 h). The mean and SE values, shown in Table 1,
show that both a and b (for
Ipeak) are significantly augmented by quinapril,
with no effect on the residual current r or on
half-inactivation voltages. The slope factor k was also
unchanged (not shown for clarity). This suggests that both a transient
and a delayed rectifier current are enhanced by ACE inhibition. We then
proceeded to investigate whether this could be correlated to an
augmentation in the expression of proteins underlying these currents.
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Table 1.
Parameters obtained from analysis of inactivation of total outward
currents using the sum of two Boltzman functions and the residual
fraction r
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The increase in current magnitude was shown earlier to be sensitive to
cycloheximide, suggesting that protein synthesis is involved. The
transient outward current in rat ventricular cells is associated with
the channel proteins Kv4.2 and Kv4.3 (14, 18, 34). The
molecular correlate of Ilate is unclear and is presumed to be a mixture of several isoforms, including Kv1.2 (34).
Nishiyama et al. (35) reported that under diabetic
conditions, there is a decrease in Kv4.2 message and protein levels. Interestingly, hypothyroid conditions also resulted in a reduction in
message levels of Kv4.2 (36). Qin et al. (39)
also showed a reduction in message and protein levels of Kv4.2 in
diabetic conditions. These changes in Kv4.2 presumably underlie the
attenuation in the transient outward current in diabetic and
hypothyroid conditions (26, 46).
We set out to establish whether the augmentation in
Ipeak by the ACE inhibitor quinapril is
paralleled by an increase in Kv4.2. We performed Western blots on
homogenized ventricular tissue obtained from hearts perfused for
7-9 h. We used control and diabetic rat hearts, with the latter
divided into untreated hearts and hearts perfused with 1 µM
quinapril. We confirmed that Kv4.2 density is attenuated under our
experimental conditions as well, although in this group the reduction
in Kv4.2 density in diabetic hearts was not significant. Nevertheless,
Kv4.2 protein in diabetic hearts was significantly (P < 0.03) augmented after perfusion with quinapril compared with
untreated diabetic hearts. Figure 2,
top left, shows sample blots showing results from the three
experimental groups. HEK-293 cells, transfected with cDNA for Kv4.2,
were also used as a control to ensure the correct identification of the
band corresponding to Kv4.2. A further control was obtained by
incubating tissue with the peptide used to generate the antibody. This
eliminated the signal at the appropriate location on the gels (Fig. 2,
top right). Densitometry scans of the gels were used to
quantify the signals, with the mean (±SE) data (normalized OD) shown
in Fig. 2, bottom.
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Fig. 2.
Western blots obtained with antibodies directed against
Kv4.2. Top: two gels from ventricular extracts obtained from
control (Cont) and streptozotocin (STZ)-treated rats in the absence of
quinapril or after 9-h perfusion with 1 µM quinapril (Q). The
left shows one lane in which extracts of human embryonic
kidney (HEK)-293 cells, transfected with Kv4.2, were run in parallel to
the ventricular extracts. The right shows that incubation
with the control peptide used to generate the antibodies (4 blank
lanes) prevents labeling of the band that corresponds to Kv4.2
(although other bands were also blocked). In each gel, extracts from 2 different rats are shown for each group. Bottom: summary
data obtained by densitometry. Optical density (OD)/100 µg protein is
plotted for the 3 groups, showing that in STZ-treated rats there is a
significant (* P < 0.03) increase in Kv4.2 density
after quinapril treatment. OD normalized to scanned bands on Coomassie
blue-stained gels gave similar results.
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This result shows that the augmentation of the transient outward
current, which occurs after a reduction in the formation of
angiotensin, is associated with an enhanced synthesis of Kv4.2 channels.
Western blot using the Kv1.2 antibody showed that this protein is
reduced in diabetic hearts compared with control and, more importantly,
that Kv1.2 is significantly (P < 0.02) augmented after
6-9 h in quinapril. This result is shown in Fig.
3. Blotting with this antibody
consistently gave two bands, which may represent two forms of the
channel (e.g., one phosphorylated). The identity of the band for Kv1.2
(used in densitometry) was confirmed in a control experiment using the
competing peptide. This abolished the lower of the two bands, as shown
in Fig. 3.
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Fig. 3.
Western blot using an antibody directed against Kv1.2.
Top: gels with the band for Kv1.2 using cardiac extracts
from control (C) rats, STZ-treated rats, and STZ-treated rats after
perfusion for 8-9 h with 1 µM quinapril (Quin). Two bands were
labeled by this antibody, but the competing peptide only blocked the
lower one, as shown in the two lanes on the right. Extracts from 2 control rats in the absence and presence of the peptide identify the
lower band as corresponding to Kv1.2. Bottom: mean ± SE values of the OD (normalized to an arbitrary band on a Coomassie
blue-stained gel with the same samples). Kv1.2 is significantly
decreased in diabetic hearts, but quinapril significantly enhances the
abundance of this protein. * P < 0.05.
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The results with Western blot show that the increase in transient and
sustained (late) currents is paralleled by an increase in channel
protein density. These results correlate to the increase we found in
parameters a and b (for the early current) in the inactivation process (see above) and thus provide strong validation for
the analysis of Himmel et al. (21), who suggested that
a and (early) b correspond to a sustained and a
transient current, respectively.
Hypothyroid conditions.
Because the local cardiac renin-angiotensin system is activated in a
variety of pathological conditions (11), we proceeded to
examine whether similar effects could be demonstrated in situations other than diabetes. We have previously extensively studied the effects
of altered thyroid status on the same K+ currents,
demonstrating a reduction in both transient and sustained currents in
hypothyroid conditions (46). Thus, in the next set of
experiments, we studied whether ACE inhibition could restore the
attenuated currents in myocytes from hypothyroid rats, as is the case
in diabetic conditions. Similar protocols were followed, with exposure
of myocytes to 1 µM quinapril for 6 h or longer. This procedure
significantly enhanced both the transient and sustained outward
currents, measured at +50 mV. Quinapril increased
Ipeak from 15.3 ± 0.8 (40 cells) to
19.7 ± 1.0 (47 cells) pA/pF (P < 0.003) and
Ilate from 5.9 ± 0.3 to 7.3 ± 0.3 pA/pF (means ± SE, P < 0.003).
Current-voltage relationships were plotted showing a significant
(P < 0.03 to P < 0.0003) enhancement
of Ipeak between 0 and 50 mV.
Ilate was significantly (P < 0.002) enhanced between 30 and +50 mV. Figure
4 shows these results. Sample current
traces are shown in Fig. 4A, and the current-voltage
relationships for Ipeak and
Ilate are shown in Fig. 4B.
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Fig. 4.
Effects of an ACE inhibitor on K+ currents in myocytes
from hypothyroid rats. A: sample current traces obtained in
response to pulses from 80 mV to potentials ranging from 0 to +50 mV
in the absence of quinapril (left) or after 6.5 h in 1 µM quinapril (right). B: current-voltage
relationships for Ipeak (left) and
Ilate (right) in the absence of
quinapril ( ; n = 27) or after 6-9
h in 1 µM quinapril ( ; n = 32). Mean
(±SE) current densities are shown. For Ipeak,
the differences were significant (P < 0.003) between
30 and +50 mV (asterisks left out for clarity). For
Ilate, the differences were significant
(P < 0.003) between 20 and +50 mV.
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Western blots of ventricular tissue from hypothyroid rats showed a
marked and significant (P < 0.007) reduction in Kv4.2. This corresponds to the reduction in mRNA levels for Kv4.2 in hypothyroid rat ventricles, as reported by Nishiyama et al.
(36). Importantly, the perfusion of hearts from
hypothyroid rats for 6 h or longer with 1 µM quinapril produced
a clear and significant (P < 0.006) increase in the
density of Kv4.2. A sample blot is shown in Fig.
5A. Figure 5B shows
the mean (±SE) ODs (normalized to protein loading levels) obtained by
densitometry in the three groups.
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Fig. 5.
A: Western blots obtained with antibodies
directed against Kv4.2. Blots were obtained from ventricular tissue in
control, hypothyroid [thyroidectomized (T)], and hypothyroid hearts
perfused for 9 h with 1 µM quinapril (2 rats from each group).
B: histogram showing mean (±SE) values of the OD/100 µg
protein for each group. Kv4.2 is significantly (P < 0.05) reduced in hypothyroid conditions. After perfusion with
quinapril, Kv4.2 density is significantly (P < 0.007)
enhanced. * P < 0.05.
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To conclude thus far, the results point to a mechanism common to both
diabetes and hypothyroid conditions, whereby local ANG II levels are
increased, with a consequent suppression of outward K+
currents. The Kv4.2 component appears to be downregulated. Blocking ANG
II formation augments both the transient current and Kv4.2 density.
Involvement of ET-1.
We next examined whether other autocrine/paracrine factors could be
implicated in suppressing the two K+ currents in the
diabetic and hypothyroid states. Of particular interest was the peptide
ET-1. This peptide, which may be involved in cardioprotection and/or
arrhythmogenesis, is released in the heart under conditions of stress
(12, 41). Importantly, there is evidence that ET-1 is
released from the myocytes themselves (1, 23). ET-1 was
also of particular interest because among its multiple actions is the
suppression of several K+ currents in rat and human
ventricular myocytes (7, 24, 28; see also DISCUSSION) as
well as an activation and selective translocation of PKC- (25,
43, 47).
Using the same rationale as with ANG II, we hypothesized that if ET-1
is released from the isolated myocytes and causes current suppression,
then a receptor inhibitor might abolish this effect. In the next set of
experiments, myocytes were exposed to the nonselective ET-1 receptor
blocker PD142893 (0.5 µM) for 6 h or longer. The blockade of
ET-1 receptors in myocytes from diabetic rats led to a significant
enhancement of both Ipeak and
Ilate. In these experiments,
Ipeak (at +50 mV) was enhanced from 16.2 ± 0.7 (n = 70) to 20.0 ± 0.7 pA/pF
(n = 68, P < 0.0004), whereas
Ilate was augmented from 5.8 ± 0.2 to
7.3 ± 0.3 pA/pF (P < 0.0004). In a smaller
number of cells, current-voltage relationships were obtained.
Ipeak was significantly augmented by PD142893
between 40 (P < 0.05) and +50 mV (P < 0.0001). Ilate was significantly elevated
between 30 (P < 0.04) and +50 mV (P < 0.002). These results are shown in Fig.
6.
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Fig. 6.
Effects of endothelin (ET)-1 receptor blockade on K+
currents in diabetic myocytes. A: sample current traces
(same voltage protocol as above) obtained from an untreated cell
(left) and from a cell incubated for 6 h with 0.5 µM
PD142893 (right), a blocker of (types A and B) ET-1
receptors. B: current-voltage relationships for
Ilate (left) and
Ipeak (right) in the absence of
PD14289 (; n = 17) or after >6-h
exposure to PD142893 (; n = 11). The
currents were significantly enhanced by PD142893 between 30 and +50
mV for Ilate and between 40 and +50 mV for
Ipeak. C: summary data for
Ilate (left) and
Ipeak (right) densities (at +50 mV)
showing increases in Ilate and
Ipeak (see text for values).
** P < 0.005.
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Myocytes from hypothyroid rats were also incubated with the ET-1
receptor blocker. Exposure to 0.5 µM PD142893 for >6 h caused an
increase in Ilate from 6.0 ± 0.2 (n = 60) to 7.91 ± 0.9 pA/pF (n = 56, P < 0.0002). The increase in
Ipeak was from 18.0 ± 0.9 to 21.6 ± 1.6 pA/pF (P < 0.05). These results (not shown)
suggest that in both diabetes and under hypothyroid conditions, there is a tonic release of ET-1 from cardiac myocytes, which contributes to
K+ current attenuation.
In further experiments, we tested whether blocking the formation of
ET-1 by inhibition of ECE would also lead to current augmentation. Cells from diabetic rats were incubated (6-8 h) in 5 µM of a
peptide fragment of Big ET-1 (16-38) that has been
shown to block ECE (32, 51). The inhibition of ECE led to
a marked and significant enhancement of both currents. At +50 mV,
Ipeak was enhanced from 14.8 ± 1.3 (n = 35) to 18.8 ± 1.1 pA/pF (n = 36, P < 0.025), whereas Ilate
was enhanced from 4.9 ± 0.2 to 7.2 ± 0.3 pA/pF
(P < 0.0001). Sample current traces and the
current-voltage relationships are shown in Fig.
7.
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Fig. 7.
Effects of inhibition of ET-converting enzyme (ECE) on outward
currents in cells from STZ-induced diabetic rats. A: sample
current traces from an untreated cell (left) and from a cell
incubated for 7 h with 5 µM of the 16-38 fragment of Big
ET-1, an ECE inhibitor (right). Current traces are in
response to 500-ms pulses from 80 mV to potentials ranging from 20
to +50 mV. B: current-voltage relationships for
Ipeak (left) and
Ilate (right) in the absence
() or presence () of the ECE inhibitor.
The effects of the ECE inhibitor were highly significant
(P < 0.001) for both currents between 20 and +50
mV.
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Thus blockading either the formation or binding of ET-1 leads to
current augmentation, strongly supporting the suggestion that ET-1 is
released by the myocytes, with a consequent suppression of these
K+ currents. We subsequently tested whether the
enhancement of the transient current could be related to an increase in
channel protein.
Hearts from thyroidectomized rats were perfused for 7-8 h with
either control solution (n = 6) or the ET-1 receptor
blocker PD142893 (0.27 µM, n = 7). Western blots from
membrane extracts from these hearts were used to assay the level of
Kv4.2. As shown in Fig. 8, the blocking
of ET-1 receptors led to a significant (P < 0.04)
enhancement of Kv4.2 density.
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Fig. 8.
Effects of blocking ET-1 receptors on the density of
Kv4.2 in thyroidectomized rats. Top: Western blot using
antibodies for Kv4.2 obtained from extracts from hearts perfused for
>7 h. The bands are from a control rat, thyroidectomized rats, and
thyroidectomized rats perfused with 0.27 µM PD142893 (missing bands
in middle were from a different protocol). Bottom: mean
normalized (to a band on a Coomassie blue-stained gel) OD values in
thyroidectomized rats in the absence (open bars; n = 6)
or presence (hatched bars; n = 7) of 0.27 µM
PD142893. The density of Kv4.2 was significantly
(* P < 0.04) enhanced by blocking ET-1
receptors.
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Involvement of PKC.
We subsequently attempted to gain further insight into the cellular
mechanisms involved in these autocrine/paracrine effects. ANG II has a
multitude of intracellular effectors (11). One target of
interest is PKC, which is a key regulator of many cellular functions
(20). It was hypothesized that if the activation of PKC by
ANG II (or by ET-1) mediates the attenuation of the K+
currents under investigation, then exposure of cells to a PKC inhibitor
would at least partially restore current density.
In the next set of experiments, myocytes from diabetic rats were
exposed to the PKC inhibitor bisindolylmaleimide. After 6-9 h of
incubation with this drug (100 nM), both Ipeak
and Ilate were significantly (P < 0.002) augmented. The mean current density of
Ipeak at +50 mV was 17.3 ± 0.8 pA/pF
(n = 51) in the absence of the PKC inhibitor and
21.7 ± 1.1 pA/pF (n = 36) after incubation with
the PKC inhibitor. The corresponding values for
Ilate were 5.0 ± 0.2 and 7.2 ± 0.4 pA/pF. These results are shown in Fig. 9.
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Fig. 9.
Effects of protein kinase C (PKC) inhibition on K+
currents in cells from STZ-induced diabetic rats. A: sample
current traces in response to pulses ranging from 10 to +50 mV shown
in the absence of inhibitor (left) or after 7-h incubation
with 100 nM bisindolylmaleimide, a PKC inhibitor. B: summary
data of Ipeak (left) and
Ilate (right) densities (at +50 mV)
in the absence of inhibitor and after 6-9 h with the PKC
inhibitor. ** P < 0.005.
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There are many isoforms of PKC, several of which are present in the
adult rat ventricle (9). Furthermore, both hypothyroid and
diabetic conditions are associated with an activation of the -isoform of PKC (29, 42). This is associated with
subcellular redistribution, with a translocation from cytosol to
membrane fractions (29, 43). Interestingly, the inhibition
of ANG II receptors was shown to prevent this redistribution in
diabetic hearts (29). We therefore sought to examine
whether maintaining PKC- in an activated/translocated state would
prevent the restoration of attenuated current magnitudes toward their
normal values.
In these experiments, we used dioctanoyl-rac-glycerol (DiC8), a
synthetic analog of diacylglycerol, the endogenous activator of (most
isoforms of) PKC (20). The incubation of cells with DiC8
was designed to maintain the activation and translocation of PKC
despite the addition of the ACE inhibitor. When cells from diabetic
rats were incubated with 20 µM DiC8 for 1 h, followed by 1 µM
quinapril for >6 h (still in the presence of DiC8), the enhancement of
Ipeak by quinapril was completely abolished,
whereas the enhancement of Ilate was partially
blocked. This result is shown in Fig.
10. Figure 10A shows current
traces obtained from cells in the absence of quinapril
(left) or after exposure to quinapril (middle) as
well as from a cell incubated with DiC8 and quinapril
(right). Figure 10B shows the current-voltage
relationships for these conditions, with mean (±SE) current densities
shown for Ipeak (left) and
Ilate (right). Note that DiC8 was
completely effective in abolishing the effect of quinapril on
Ipeak but was only partially effective in
reducing the effect on Ilate.
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Fig. 10.
Effects of quinapril in the presence of a diacylglycerol analog.
A: current traces (in response to steps from 80 mV to
potentials ranging from 10 to +50 mV) obtained in cells from diabetic
rats. The traces shown are from cells without quinapril
(left), after 6 h in 1 µM quinapril
(middle), and after 6 h in quinapril and 20 µM
dioctanoyl-rac-glycerol (DiC8; added 1 h before quinapril;
right). B: current-voltage relationships for
Ipeak (left) and
Ilate (right) corresponding to the 3 experimental conditions. Mean ± SE values are shown. DiC8
completely prevented the enhancement of the transient current by
quinapril but only partially prevented the effect on
Ilate.
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Similar results were obtained with myocytes from hypothyroid rats. The
current densities (at +50 mV) for Ipeak in the
absence of quinapril, after 6-9 h in quinapril, and after 6-9
h in quinapril and DiC8 were 17.1 ± 1.1 (n = 29),
23.9 ± 1.0 (n = 28), and 17.9 ± 1.1 pA/pF
(n = 25). The corresponding values for
Ilate in these cells were 5.6 ± 0.2, 8.2 ± 0.2, and 5.9 ± 0.3 pA/pF. The increase by quinapril
was highly significant (ANOVA) for both currents (P < 0.001), as was the attenuation of currents by the combination of DiC8
and quinapril (P < 0.001). The attenuation of
Ilate was more complete in cells from
hypothyroid than diabetic rats. The reason for this is unclear at this stage.
We next considered whether the effect of maintained PKC
activation/translocation on current magnitude was correlated to effects on channel protein. The experiments were repeated by perfusing hearts
from hypothyroid and diabetic rats with quinapril alone (for >7 h) or
with DiC8 for 0.5 h, followed by quinapril and DiC8 for >7 h.
Subsequent Western blots using the Kv4.2 antibody showed that the
enhancement of Kv4.2 density by quinapril (as shown in Figs. 2 and 5)
was abolished when DiC8 was present. These results are shown in Fig.
11. Figure 11, left, shows
data from hypothyroid rats, and Fig. 11, right, shows data
from diabetic rats.
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Fig. 11.
Enhancement of Kv4.2 density by ACE inhibition is blocked by PKC
activation. Top left: Western blots with bands obtained from
a control rat, 2 thyroidectomized rats, 3 thyroidectomized rats
perfused with 1 µM quinapril for >7 h, and 3 thyroidectomized rats
perfused (7 h) with quinapril and 20 µM DiC8 (added 30 min before
quinapril). Right, bands from 2 STZ-induced diabetic rats, 3 STZ-induced diabetic rat hearts perfused with quinapril for 8-9 h,
and 3 bands from diabetic hearts perfused with quinapril and DiC8. The
augmentation of Kv4.2 density by quinapril in both hypothyroid and
diabetic conditions is clearly blocked by DiC8; 100 µg protein was
loaded in all lanes. Bottom: summary data (means ± SE), with band densities normalized to an arbitrary band on a Coomassie
blue-stained gel, obtained in parallel to the Western blot.
Left, data from hypothyroid hearts (n = 8, 0.06 < P < 0.07); right, data from
diabetic hearts (n = 3, P < 0.05).
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ANOVA showed that (for this small sample) the reduction of channel
density was significant (P < 0.05) for diabetic hearts and was not quite significant (0.06 < P < 0.07)
for hypothyroid rats. However, these results are completely consistent
with the highly significant effects of this protocol on the
K+ currents in diabetic and hypothyroid conditions.
DiC8 may activate several PKC isoforms and may also have other
PKC-independent effects as well (6). To more specifically implicate the intracellular translocation of PKC-, we took advantage of recent studies that have investigated the intracellular movement of
PKC from its cytosolic to its membrane binding sites (31). Specific peptides have been designed to activate or inhibit the translocation of specific PKC isoforms (10). In the
following experiments, we incubated cells from diabetic rats with
specific peptides linked to a carrier (antennapedia) that renders them membrane permeable (8). We then added the ANG II receptor
blocker saralasin, which was shown to augment
Ipeak and Ilate
(45). Initially, we incubated cells with 1 µM of a
specific agonist of PKC- translocation (10) for 1 h. Saralasin was then added for >6 h, and currents were measured. As
with quinapril and DiC8, the persistent activation of PKC translocation
prevented the restoration of Ipeak and
Ilate by saralasin. Figure
12 illustrates this result. Figure
12A shows current traces from diabetic myocytes exposed for
7 h to 1 µM saralsin either in the absence (left) or
presence of the PKC- agonist (right). Figure
12B shows the mean current densities at +50 mV for
Ipeak (left) and
Ilate (right) in the absence (90 cells) and presence of saralasin with (85 cells) and without (86 cells)
the PKC- agonist. The corresponding values for
Ipeak are 18.6 ± 0.8, 22.8 ± 0.9, and 18.7 ± 0.8 pA/pF and for Ilate are
6.0 ± 0.2, 7.9 ± 0.3, and 6.6 ± 0.2 pA/pF. When PKC
is maintained in its activated/translocated state, saralasin can no
longer augment either current (P < 0.0003).
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Fig. 12.
Effects of the ANG II receptor blocker saralasin in the presence
of a specific PKC- translocation agonist peptide. A:
current traces obtained in cells from diabetic rats after incubation
for 7 h with 1 µM saralasin (left) or with 1 µM
saralasin and 1 µM peptide added 1 h before saralasin
(right). B: summary data showing mean (±SE)
current densities (at +50 mV) of Ipeak
(left) and Ilate (right)
in the absence of saralasin, in the presence of saralasin, and with
saralasin and the PKC- agonist peptide. * P < 0.01; ** P < 0.001.
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We also tested whether this effect was specific to the -isoform of
PKC by incubating cells with the translocation agonist peptide specific
for the -isoform (10). The PKC- translocation agonist peptide was added (1 µM) to the incubation medium for 1 h, followed by 1 µM saralasin for >6 h. In contrast to the PKC- translocation agonist peptide, the peptide specific for the -isoform did not abolish the effects of saralasin. Ipeak
and Ilate were both significantly
(P < 0.0001) augmented by saralasin in the presence of
this peptide. At +50 mV, Ipeak increased
from 13.5 ± 1.0 (n = 44) to 19.2 ± 1.3 pA/pF (n = 36), whereas Ilate
increased from 4.7 ± 0.2 to 6.5 ± 0.3 pA/pF. A control
dimer of the antennapedia carrier used to transport these peptides into
the cells was also without effect, enabling saralasin to significantly
(P < 0.0001) enhance Ipeak and
Ilate. In this group,
Ipeak was increased from 15.6 ± 1.0 (n = 32) to 21.8 ± 1.2 pA/pF (n = 27), and Ilate was increased from 5.6 ± 0.2 to 7.6 ± 0.3 pA/pF.
These results suggest that PKC- redistribution towards the normal
prediabetic state is a prerequisite for K+ current augmentation.
Because ET-1 is also an activator of PKC (12), and
of PKC- in particular (43, 47), we also examined
whether a maintained activation of PKC- with the translocation
activator would prevent the enhancement of K+ currents by
ET-1 receptor blockade. In this set of experiments, 0.5 µM PD142893
(>6-h incubation) augmented Ipeak from
17.7 ± 0.9 (n = 47) to 21.6 ± pA/pF
(n = 44, P < 0.05). However, when 1 µM of the PKC- translocation activator was added 1 h before PD142893, this augmentation was blunted, with a mean density (at +50
mV) of 18.1 ± 0.9 pA/pF (n = 44), which is
significantly (P < 0.05) smaller than with PD142893
alone. The corresponding values for Ilate were
5.7 ± 0.2, 7.5 ± 0.4, and 7.0 ± 0.3 pA/pF. As with ANG II, the augmentation of Ilate by ET-1
blockade is only slightly reduced by maintaining PKC in an activated
state (see DISCUSSION).
Control myocytes.
It has been suggested that some of these autocrine/paracrine factors
may be released under basal conditions, with an increase occurring
under stress situations such as ischemia (12). In the final set of experiments, we tested whether quinapril or PD142893 would affect these K+ currents in cells from control rats.
We found that an exposure of 6-9 h of cells from control rats to
either 1 µM quinapril (21 cells) or 0.5 µM PD142893 (23 cells)
significantly augmented Ilate but had no effect
on Ipeak. In control cells (n = 24), the mean densities (at +50 mV) for Ipeak
and Ilate were 22.8 ± 1.3 and 6.2 ± 0.3 pA/pF, respectively. The corresponding values after ACE inhibition
by quinapril were 20.1 ± 1.5 (P > 0.05) and
7.3 ± 0.3 pA/pF (P < 0.02). After exposure to
the ET-1 receptor inhibitor, the mean densities were 21.3 ± 2.1 (P > 0.05) and 7.4 ± 0.2 pA/pF (P < 0.003).
| |
DISCUSSION |
Summary of results.
The results presented above provide several novel findings. Transient
and sustained K+ currents in rat ventricular myocytes are
attenuated under both diabetic and hypothyroid conditions. We showed
previously that these currents are augmented after the inhibition of
ANG II action or formation in diabetic conditions (Fig. 1). We now show
that this is the case in hypothyroid rats as well (Fig. 4) and
demonstrate that the enhancement of the transient current involves an
augmentation of Kv4.2, one of the channel proteins underlying this
current (Figs. 2 and 5). In diabetic conditions, we also showed (Fig. 3) that quinapril augments Kv1.2, which underlies some of
Ilate in rat ventricular myocytes
(34). Furthermore, we found that under diabetic and
hypothyroid conditions, an autocrine/paracrine release of ET-1
modulates these K+ currents. The in vitro blockade of ET-1
receptors or the inhibition of ET-1 formation in myoctes from diabetic
or hypothyroid rats also leads to enhancement of these K+
currents (Figs. 6 and 7) associated with enhanced Kv4.2 protein (Fig.
8). The results also show that part of the mechanism(s) mediating the
changes in current density by both ANG II and ET-1 depend(s) on a
subcellular redistribution of PKC-. The attenuation of
K+ currents is mediated by PKC activation, because a PKC
inhibitor can reverse this attenuation (Fig. 9). This augmentation
apparently depends on a recovery of PKC- distribution from the
pathological toward the normal pattern (Figs. 10-12). Thus
maintaining PKC- in an activated/translocated state prevents the
enhancement of Ipeak by ANG II and ET-1
inhibition. The enhancement of Ilate is
apparently less affected by PKC-related mechanisms than
Ipeak. Further work is required to establish the
contribution of Kv1.2 to the b component of
voltage-dependent inactivation of Ipeak.
Interpretation and significance.
The simplest interpretation of these results is consistent with the
reported activation of the local cardiac renin-angiotensin system
(11) and the autocrine release of ANG II by the myocytes or a paracrine release from nonmyocytes. In parallel, or as a result of
ANG II action (1, 23), there is also an activation and
autocrine/paracrine release of ET-1. Both ANG II and ET-1 contribute to
suppression of the transient and sustained outward currents, the major
repolarizing K+ currents in ventricular cells. The
suppression of K+ currents by exogenous ET-1 when added
acutely in vitro has been shown previously (7, 24). We
have shown (44) that an acute activation of PKC by DiC8,
which normally suppresses K+ currents, does not have this
action in diabetic and hypothyroid conditions, presumably because PKC
is already activated in these pathologies. Thus acute actions of ET-1
released by autocrine mechanisms would presumably not be present
(although this was not tested directly). Our present data suggest an
endogenous chronic release under diabetic and hypothyroid conditions
that attenuates currents at least partially by inhibiting synthesis of
the channel -subunits.
In combination, ANG II and ET-1 action on K+ currents will
produce action potential prolongation (45) that may
underlie the prolongation of the Q-T interval in the electrocardiogram,
often recorded in diabetes and hypothyroidism (2, 15). The
restoration of current magnitudes takes several hours and depends on
protein synthesis, with a concomitant increase of Kv4.2, a channel
protein underlying the transient current. This suggests that ANG II and ET-1 repress the synthesis of (at least some of the) channel proteins underlying this current. When the autocrine action is blocked, channel
protein synthesis can return to normal levels.
In addition, a key role for PKC- is suggested by the present work.
When PKC- activation/translocation is maintained, the enhancement of
Ipeak by ANG II or ET-1 blockade in both
diabetic and hypothyroid conditions is abolished (Figs. 10 and 12).
However, the enhancement of Ilate is only
partially affected by maintaining PKC activation. Kv4.2 enhancement by
ACE inhibition is also prevented by PKC activation by DiC8 (Fig. 11).
The working hypothesis based on these results is that on activation,
some of the translocation of PKC- is to the nucleus. PKC is involved
in gene regulation by activation or binding of various transcription
factors (20). PKC- may thus directly suppress the
synthesis of channel proteins underlying Ipeak
but only some of the proteins underlying Ilate. There is abundant evidence showing that under diverse pathologies, there is an increase in PKC activation along with an attenuation of
Ipeak. This occurs, for example, in cardiac
hypertrophy (17, 30), hypoxia (16), or
hypothyroid conditions (42, 46). The prevention of
enhanced Kv4.2 synthesis by DiC8 does not positively confirm a causal
connection between PKC activation and attenuation of channel synthesis,
but this is a highly likely interpretation. It is of interest, however,
that maintaining the activation of PKC only partially reverses the
effects of ANG II and ET-1 inhibition on the noninactivating outward
current (Fig. 10 and results with ET-1 blockade in the presence of the
PKC- translocation activator). This implies that the enhancement of
this current is achieved partially through a PKC-independent mechanism.
Because the sustained outward current expresses a mixture of several
channels (21, 34), it is possible that the enhancement of
one component of Ilate is PKC dependent, whereas
others are augmented by a PKC-independent mechanism. The different
protocols showed variability in the degree of suppression of
Ilate by maintained PKC activation. This issue will require further investigation.
The results presented here are significant on several levels. First,
they expand previous work on autocrine/paracrine mechanisms in the
heart, showing that multiple agents are released. It is presumed that
these agents are released from the myocytes themselves, because these
are the dominant cells present. However, we cannot rule out release by
other (nonmyocyte) cells obtained in the enzymatic dispersion process.
Furthermore, it has been suggested that ET-1 release is activated by
ANG II (1, 23). The present experiments cannot distinguish
between this possibility and an independent release of ET-1. The
interaction between the two signaling pathways in the regulation of
K+ currents is obviously complex and will need further
investigation. There may also be some basal release from control
myocytes, as suggested previously (12). Blocking ACE or
ET-1 receptors in control cells augmented Ilate,
suggesting a tonic suppression of this current.
Ipeak was unaffected, suggesting that only under pathological conditions is this current affected by autocrine mechanisms.
The present work establishs that effects on K+ currents,
presumably reflecting autocrine release, occur under diverse
pathologies. This will impact cardiac electrical activity, because the
attenuation of cardiac K+ currents will prolong the action
potential and potentially contribute to arrhythmogenesis (19,
27). This could occur by an alteration of repolarization
patterns (48) or by appearance of early
afterdepolarizatons and indirect changes in calcium influx
(37). Diabetes and hypothyroidism are often accompanied by
increased susceptibility to cardiac arrhythmias (5, 13,
15). Thus the proven benefits to diabetic patients receiving ACE
inhibitors (52) may be related to a reduction in cardiac
arrhythmias in addition to the alleviation of hypertension, as we
suggested earlier (45). On the basis of present results, the benefits of long-term ET receptor blockade in diabetes
(49) may also extend to protection from arrhythmias.
Limitations of study.
There are several limitations to this study, although these were
considered not to affect the major conclusions. First, it was beyond
the scope of the work to directly measure the release of ANG II and
ET-1 from the myocytes to establish whether there are true autocrine
mechanisms involved. However, such release has been demonstrated
previously (1, 11). The combined use of multiple
interventions (saralasin, quinapril, PD142893, and the ECE inhibitor in
both diabetic and hypothyroid conditions) strongly supports our working
hypothesis and rules out possible nonspecific effects of any one of the
agents used. It was beyond the scope of this work to fully analyze
changes in other channel proteins. However, the changes measured in
Kv4.2 and Kv1.2 strongly support the working hypothesis, whereby ANG II
and ET-1 suppress channel protein synthesis. The signals for Kv4.3 were
considerably less satisfactory despite the use of two different
antibodies and different methods of membrane purification. There was
some disadvantage to the Western blotting experiments in that
ventricular tissue rather than isolated cells were used. This, however,
is common practice (e.g., 36) and was done here to obtain sufficient protein. Although other cell types are abundant in ventricular tissue,
the predominant tissue mass is provided by the myocytes. Other cells
such as fibroblasts lack Kv4.2. In addition, the parallel changes in
Ipeak and Kv4.2 that were obtained also strongly
support our conclusions. A final limitation lies in the fact that no
direct evidence was obtained for a maintained pathological subcellular distribution of PKC with DiC8 and the peptide agonists. This too was
beyond the scope of the present work, but it should be emphasized that
Malhotra et al. (29) showed that ANG II receptor blockers prevented PKC- redistribution in cardiac cells, consistent with our findings.
| |
ACKNOWLEDGEMENTS |
This study was supported by a grant from the Canadian Diabetes
Association in honour of the late Celeste DePaoli and by a grant from
the Heart and Stroke Foundation of Alberta, Canada.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Shimoni, Dept. of Physiology and Biophysics, Health Sciences Centre, 3330 Hospital Dr., NW, Calgary, Alberta, Canada T2N 4N1 (E-mail: shimoni{at}ucalgary.ca).
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.
10.1152/ajpheart.00748.2002
Received 28 August 2002; accepted in final form 13 December 2002.
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TOP
ABSTRACT
INTRODUCTION
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