The Cardiovascular Institute and Departments of 1Medicine and 2Physiology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153
Submitted 2 October 2002 ; accepted in final form 25 February 2003
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ABSTRACT |
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heart; signal transduction; hypertrophy; transcription; mRNA stability; sarco(endo)plasmic reticulum Ca2+-ATPase
Cultured neonatal rat ventricular myocytes (NRVM) have proven to be useful tools in understanding the cellular mechanisms regulating SERCA2 gene expression in response to neurohormonal and mechanical stimuli. For instance, previous studies have shown that SERCA2 gene expression is regulated by peptide growth factors (33), thyroid hormones (24), angiotensin II (28), endothelin-1 (ET) (23), and norepinephrine (9). Studies from our laboratory have shown that mechanical loading of NRVM reduced SERCA2 mRNA and protein levels compared with unloaded cells (4, 8). Importantly, reduced SERCA2 gene expression was reflected functionally by a significant prolongation of the intracellular Ca2+ concentration ([Ca2+]i) transient (4) and a significant reduction in SR pump activity (8) in cellular homogenates of mechanically loaded cells.
Several groups have also begun to evaluate the intracellular signaling
pathways that regulate SERCA2 gene expression. At present, the most compelling
evidence suggests that the Ras-Raf-MEK-ERK cascade is both necessary and
sufficient to downregulate SERCA2 and that one or more isoenzymes of protein
kinase C (PKC) may also be involved
(27). Indeed, PKC activation
was necessary for Ras-GTP loading in response to the hypertrophic agonists
phenylephrine and ET (13), and
phorbol 12-myristate 13-acetate (PMA), a direct activator of the conventional
and novel PKCs, induced Ras activation via stimulation of guanine nucleotide
exchange (31). PMA also
markedly downregulated SERCA2 mRNA levels in NRVM
(21,
23,
36), which could be prevented
by the nonselective, PKC inhibitors staurosporine and chelerythrine
(36). However, NRVM express
three different phorbol ester-sensitive PKC isoenzymes (PKC, PKC
,
and PKC
) (38), which
may be differentially regulated and have specific functions in the
cardiomyocyte (26). This
specificity is likely due to their differential activation by hypertrophic
stimuli (5,
16,
35,
43) and their differential
localization within the cell
(19). Nevertheless, the role
of each PKC isoenzyme in the regulation of SERCA2 gene expression remains
unknown. Therefore, in this study we have used adenoviral vectors encoding
wild-type, constitutively active, and kinase-defective forms of PKC
,
PKC
, and PKC
to analyze their individual effects in regulating
SERCA2 gene expression in NRVM.
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MATERIALS AND METHODS |
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Cell culture. Animals used in these experiments were handled in
accordance with the "Guiding Principles in the Care and Use of
Animals," approved by the Council of the American Physiological Society.
Ventricular myocytes were isolated from the hearts of 2-day-old Sprague-Dawley
rats by collagenase digestion, as previously described
(40). Myocytes were preplated
for 1 h in serum-free PC-1 medium to reduce nonmyocyte contamination. The
nonadherent NRVM were then plated at a density of 1,600 cells/mm2
onto collagen-coated 35- or 60-mm dishes and left undisturbed in a 5%
CO2 incubator for 1418 h. Unattached cells were removed by
aspiration and washed twice in HBSS, and the attached cells were maintained in
a solution of DMEM/medium 199 (4:1) containing antibiotic/antimycotic
solution. Under these highdensity culture conditions, NRVM displayed
synchronous [Ca2+]i transients and beating
activity (100150 beats/min) within 24 h of plating. Cardiomyocytes
were infected (60 min, 25°C with gentle agitation) with
replication-defective adenoviruses (Adv) diluted in DMEM/medium 199. This
medium was then replaced with virus-free DMEM/medium 199, and the cells were
cultured for an additional 4872 h.
Adenoviral constructs. Replication-defective adenoviruses encoding
wild-type (wt) bovine PKC, rat PKC
, and rat PKC
were
constructed by first subcloning their respective cDNAs (kindly provided by
Drs. Peter Parker and Peter Sugden, Imperial College of Science Technology and
Medicine, Cambridge, UK) into pAC-CMV-pLpA-SR plasmid. The subcloned
constructs were cotransfected along with pJM17 plasmid that contained
adenoviral DNA into HEK-293 cells. After homologous recombination, the
adenoviruses were plaque-purified, amplified by sequential infection of
HEK-293 cells, and purified by double CsCl ultracentrifugation.
Replication-defective adenoviruses encoding constitutively active (ca)
PKC
and PKC
were generated as previously described
(26,
42). Replication-defective
adenoviruses encoding dominant negative (dn) mouse PKC
(10), rat PKC
(10), and rabbit PKC
(34) were obtained from Drs.
Trevor Biden (Garvan Institute of Medical Research, St. Vincents Hospital,
Darlinghurst, Sydney, Australia) and Peipei Ping (Dept. of Physiology, Univ.
of California, Los Angeles, CA) and were amplified and purified. Finally,
replication-defective adenoviruses encoding cytoplasmic (cyto) or
nuclear-encoded (ne)
-galactosidase (
gal) were used to control for
nonspecific effects of adenoviral infection
(20). The multiplicity of
viral infection (MOI) of each adenovirus was determined by dilution assay in
HEK-293 cells.
Western blotting. NRVM were homogenized in lysis buffer
(41). Equal amounts of
extracted proteins (50 µg) were separated on 10% SDS-polyacrylamide gels
with 5% stacking gels. Proteins were electrophoretically transferred to
nitrocellulose membranes, and the Western blots were probed with antibodies
specific for PKC, PKC
, and PKC
. Primary antibody binding
was detected with horseradish peroxidase-conjugated goat anti-mouse secondary
antibody and visualized by enhanced chemiluminescence (ECL: Amersham).
mRNA analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (14) or by using the RNeasy mini kit (Qiagen, Valencia, CA). RNA was quantified by absorbance at 260 nm, and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. Both extraction methods produced similar yields of undegraded, purified RNA. SERCA2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs were then analyzed by either Northern blotting (8) or real-time RT-PCR. For Northern blots, equal amounts of total RNA (10 µg) were separated by denaturing agarose gel electrophoresis. Blots were then sequentially hybridized with a 2.3-kb cDNA probe specific for rat SERCA2, kindly provided by Dr. Wolfgang Dillmann (University of California, San Diego), and a 1.2-kb cDNA probe specific for human GAPDH, clone pHcGAP (46) obtained from the American Type Culture Collection (Rockville, MD). In some Northern blotting experiments, the amounts of SERCA2 mRNA were quantified by scintillation spectroscopy (Instant Imager, Hewlett-Packard) and expressed relative to the amounts of GAPDH mRNA in each sample. To ensure equal loading of the gels, and also to verify that the various interventions had no effect on the "bystander transcript" GAPDH mRNA used in the real-time RT-PCR assay, some Northern blots were also probed with a 32P-labeled oligonucleotide probe specific for 18S rRNA (8).
For the real-time RT-PCR, cDNA was reverse-transcribed from the extracted RNA from a reaction mixture consisting of 5x First Strand buffer (5 µl), 0.1 M DTT (2 µl), pd(N)6 random hexamers (2 µl), 10 µM dNTPs (2 µl), RNase inhibitor (1 µl), and reverse transcriptase (1 µl). A volume equivalent to 1 µg of RNA was used per sample, and RNase-free diethyl pyrocarbonate (DEPC)-treated water was added to bring the final volume of the reaction mixture to 20 µl. The sample was heated to 42°C for 30 min, followed by 95°C for 5 min. The resultant cDNA was then stored at 80°C. All real-time PCR was performed with a Bio-Rad iCycler iQ Multi-Color real-time PCR detection system. Sample 96-well plates were loaded with 50 µl of reaction mixture per well. The mixture consisted of 1 µl of sample DNA, 21 µl of DEPC water, 25 µl of Platinum Quantitative PCR SuperMix-UDG, and 3 µl of a primer/dual-labeled probe combination specific for each gene of interest. TaqMan rodent GAPDH control reagents were obtained from Applied Biosystems (Foster City, CA). For the rat SERCA2 cDNA, the following primers were used: 5'-TCT GTC ATT CGG GAG TGG GG-3' and 5'-GCC CAC ACA GCC AAC GAA AG-3'. The rat SERCA2 cDNA fluorescent probe consisted of the following sequence: 5'-TGG CCA CTC ATG ACA ACC CG-3'.
Probes were labeled at the 5'-end with 6-carboxyfluorescein (6-FAM)
and at the 3'-end with BHQ-1. Rat SERCA2 primer concentrations were 10
µM, and the probe concentration was 1 µM. Rodent GAPDH primer
concentrations were 10 µM, and the probe concentration was 5 µM. PCR
amplification was performed by cycling between 95 (15 s) and 60°C (60 s)
for 45 cycles, using the 6-FAM fluorophore for quantification. All samples
were run in triplicate, and the results were averaged. After the PCR, mRNA
levels were expressed in threshold cycles (Ct) by the iCycler. The average Ct
was then converted into an "input amount" by using a standard
curve derived from serial dilutions of SERCA2 or GAPDH cDNA. For each plate in
the experiment, the input amount for the gene of interest was standardized
first to rat GAPDH mRNA and then to the appropriate adenovirus control
(Adv-negal for the wtPKC overexpression studies, Adv-cyto
gal for
the dnPKC overexpression studies) derived from the same experiment.
Data analysis. Results are expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared by 1-way blocked analysis of variance (ANOVA) or 1-way blocked ANOVA on ranks followed by Dunnett's test, where appropriate. Data from two groups were compared by paired t-test or Wilcoxon signed rank test where appropriate. Differences among means were considered significant at P < 0.05. Data were analyzed using the SigmaStat statistical software package (ver. 1.0; Jandel Scientific, San Rafael, CA).
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RESULTS |
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We next examined the effects of Adv-mediated overexpression of each wtPKC
on the expression levels of all of the phorbol ester-sensitive PKC isoenzymes
expressed in NRVM. As shown in Fig.
1B, Adv-wtPKC (25 MOI, 48 h) increased expression
levels of PKC
10- to 20-fold but had no significant effect on the
expression of endogenous PKC
or PKC
. Similarly, Adv-wtPKC
(25 MOI, 48 h) markedly increased PKC
levels but also substantially
increased PKC
in NRVM. Endogenous PKC
levels, however, were
unaffected by wtPKC
overexpression. In marked contrast, overexpression
of wtPKC
(25 MOI, 48 h) substantially decreased endogenous PKC
and PKC
levels compared with cells infected with Adv-ne
gal.
PKC
, PKC
, and PKC
levels in Adv-ne
gal-infected NRVM
(25 MOI, 48 h) were all similar to those observed in uninfected control NRVM
(data not shown), indicating that adenovirus infection alone was not
responsible for these changes.
Overexpression of the novel PKC isoenzymes downregulates SERCA2 mRNA
levels. We then examined the effects of ET, PMA, and the individual
Adv-wtPKCs on SERCA2 mRNA levels. As previously described
(36), activation of all three
phorbol ester-sensitive PKC isoenzymes with PMA (200 nM, 48 h) substantially
reduced SERCA2 mRNA levels as assessed by Northern blotting
(Fig. 2A). ET (100 nM,
48 h), a potent activator of PKC, and to a lesser extent, PKC
(16), also caused SERCA2
downregulation, although its effect was somewhat less than that observed with
PMA (Fig. 2B). Neither
agent substantially affected the expression of GAPDH mRNA, because the
expression of this transcript did not systematically change relative to the
amount of total RNA or 18S rRNA.
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We next evaluated the effects of the Adv-wtPKCs on SERCA2 mRNA levels.
First, we found that the control adenovirus Adv-negal (25 MOI, 48 h) had
no significant effect on SERCA2 mRNA levels compared with uninfected NRVM,
indicating that adenovirus infection alone at this MOI did not produce
nonspecific effects on SERCA2 gene expression (data not shown).
Adv-wtPKC
(25 MOI, 48 h) was also without effect and in some
experiments actually appeared to increase SERCA2 mRNA levels compared with
Adv-ne
gal (Fig.
2C). In contrast, Adv-wtPKC
(25 MOI, 48 h), and to
a lesser extent, Adv-wtPKC
(25 MOI, 48 h), reduced steady-state levels
of SERCA2 mRNA. None of the adenoviruses substantially affected the expression
of GAPDH mRNA, because the expression of this transcript did not
systematically change relative to the amount of total RNA or 18S rRNA.
To obtain a more sensitive and precise assessment of these relatively
modest changes in SERCA2 mRNA, the effects of each adenovirus on SERCA2 mRNA
levels (relative to the invariant GAPDH mRNA) were then quantitatively
analyzed by real-time RT-PCR, and the results are depicted in
Fig. 3. Adv-wtPKC had no
significant effect on SERCA2 mRNA levels compared with NRVM infected with
Adv-ne
gal. However, wtPKC
and wtPKC
overexpression reduced
SERCA2 mRNA levels to 69 ± 7 and 61 ± 9% of control levels,
respectively (P < 0.05 for each adenovirus; n = 9
experiments). The overall reduction in SERCA2 mRNA levels by overexpression of
each novel PKC isoenzyme (3040%) was somewhat less than the level of
downregulation observed with PMA (5560% reduction)
(36) but was similar to that
observed with ET (23)
(Fig. 2B). Thus
overexpression of either novel wtPKC was sufficient to downregulate SERCA2
gene expression in NRVM.
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As a further check on the effects of PKC and PKC
overexpression on SERCA2 mRNA levels, NRVM were also infected (10 MOI, 48 h)
with replication-defective adenoviruses expressing constitutively active
mutant forms of these isoenzymes. [PKC
and PKC
were rendered
constitutively active by an amino acid deletion or a point mutation within
their respective pseudosubstrate domains
(49).] As shown in
Fig. 4, overexpression of
either novel caPKC isoenzyme was also sufficient to downregulate SERCA2 mRNA
levels compared with uninfected or Adv-ne
gal-infected cells.
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Effects of Adv-dnPKC, Adv-dnPKC
, and
Adv-dnPKC
on PKC levels. Initial experiments were also
conducted to ascertain the effects of adenovirus-mediated overexpression of
each dnPKC on the expression levels of all of the phorbol ester-sensitive PKC
isoenzymes. As shown in Fig. 5,
Adv-dnPKC
(100 MOI, 72 h) increased immunoreactive PKC
but also
reduced expression levels of endogenous PKC
, and to a much lesser
extent, PKC
. Similarly, overexpression of dnPKC
(100 MOI, 72 h)
reduced endogenous PKC
expression but did not substantially affect
endogenous PKC
. Finally, overexpression of dnPKC
(10 MOI, 72 h)
increased the amount of immunoreactive PKC
in NRVM but did not appear
to substantially affect either endogenous PKC
or PKC
levels.
[Reduced MOI for dnPKC
was used in this and subsequent experiments
because higher concentrations caused cell detachment within 4872 h,
presumably as a consequence of apoptosis
(22,
26).] As was noted above for
Adv-ne
gal, Adv-cyto
gal (10100 MOI, 72 h) also had no
apparent effect on endogenous PKC
, PKC
,orPKC
expression
compared with uninfected control NRVM (data not shown).
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Effects of Adv-dnPKC, Adv-dnPKC
, and
Adv-dnPKC
on SERCA2 mRNA levels. As shown in
Fig. 6, analysis of SERCA2 mRNA
levels by Northern blotting indicated that overexpression of dnPKC
(100
MOI, 72 h), dnPKC
(100 MOI, 72 h), or dnPKC
(10 MOI, 72 h)
appeared to increase SERCA2 mRNA levels compared with NRVM infected with
Adv-cyto
gal (100 MOI, 72 h). However, PMA (200 nM) added to the culture
medium 24 h after adenovirus infection was still capable of downregulating
SERCA2 mRNA in NRVM infected with each adenovirus. SERCA2 mRNA levels were
then quantitatively analyzed by real-time RT-PCR, and the results are depicted
in Fig. 7. All three dnPKCs
significantly increased SERCA2 mRNA levels over time (dnPKC
>
dnPKC
> dnPKC
). Adv-dnPKC
produced the largest increase
in SERCA2 gene expression (2.8 ± 1.0-fold; n = 11
experiments). As shown by Northern blotting, however, PMA treatment was still
sufficient to significantly downregulate SERCA2 mRNA levels despite
overexpression of each dominant negative mutant.
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DISCUSSION |
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Previous studies have demonstrated that NRVM stimulated with hypertrophic
agonists (9,
23,
28,
32), PMA
(21,
23,
36), or cyclic mechanical
loading (8) undergo
hypertrophy, and this growth response was accompanied by reduced SERCA2 gene
expression. Conversely, we previously showed that mechanical unloading
produced cardiomyocyte atrophy, which was accompanied by significant
upregulation of SERCA2 mRNA and protein levels
(4). One question raised by
these studies was whether SERCA2 gene expression could be regulated
independently of the hypertrophic response. However, we and others recently
showed that adenovirally mediated overexpression of either PKC
(7,
26,
42) or PKC
(7,
26) was not sufficient to
induce cardiomyocyte hypertrophy but, as demonstrated in the present report,
was sufficient to downregulate SERCA2 mRNA levels. Conversely, overexpression
of PKC
was sufficient to induce many features of NRVM hypertrophy in
the absence of other stimuli
(7) but was not sufficient to
downregulate SERCA2 mRNA levels. These data therefore provide new evidence for
isoenzyme-selective regulation of SERCA2 gene expression by PKC and also
indicate that this regulation can occur independently of generalized effects
on NRVM growth or atrophy. Nevertheless, a limitation of all of these
adenoviral studies (including our own) is the lack of a documented
dose-response relationship between levels of PKC overexpression, their impact
on isoenzyme activation and translocation, and their effects on cardiomyocyte
gene expression and growth. This is of some concern, because overexpressing
PKC isoenzymes at different levels could conceivably trigger dichotomous
phenotypes.
Our data demonstrating that both ET and PMA significantly reduced SERCA2
mRNA levels imply that these ET- and PMA-dependent effects were mediated by
the activation of endogenous PKC and/or PKC
. However, chronic
exposure to phorbol esters is well known to induce activation, followed by
downregulation of PKCs by intracellular proteolysis. Indeed, we demonstrate in
Fig. 1A that chronic
exposure to PMA substantially reduced endogenous PKC
, PKC
, and
PKC
during 48 h of continuous exposure to the drug. However, in our
previous paper (36), we
compared SERCA2 mRNA levels in NRVM stimulated with PMA for only 30 min vs.
cells that were treated continuously for up to 48 h and showed that continuous
exposure to PMA was not necessary to reduce SERCA2 mRNA. In fact, the stimulus
for SERCA2 mRNA downregulation was generated within 30 min of PMA exposure,
but the reduction in SERCA2 mRNA took 1224 h to become manifested.
These results are consistent with a PKC-dependent signaling pathway in which
only transient activation of PKC
and/or PKC
is required to
initiate a cascade of events that ultimately leads to reduced SERCA2 mRNA. In
other words, there must be a temporal element in the PKC stimulatory pathway
that persists even after the PKC isoenzymes are downregulated during chronic
PMA exposure. In further support of this conclusion is the observation that
although ET acutely activated PKC
and PKC
, chronic ET treatment
only partially reduced PKC
and PKC
protein levels over 48 h. It
is worthwhile to mention, however, that agonist-induced reductions in
individual PKC isoenzyme levels are best detected when protein samples are
partitioned between soluble and particulate fractions, as shown by Sabri et
al. (39).
Similarly, the specificity of Adv-wtPKC overexpression may have also been
affected by the various compensatory changes in endogenous PKC isoenzyme
expression that occurred over time. As demonstrated in
Fig. 1B, adenovirally
mediated wtPKC overexpression markedly reduced the expression levels of
endogenous PKC
and PKC
. Furthermore, we have previously shown
that overexpression of caPKC
increased the expression of endogenous
PKC
, whereas overexpression of caPKC
markedly reduced PKC
levels (26). In addition,
PKC
and PKC
activities are also regulated by transphosphorylation
within their respective hydrophobic and activation loop domains, providing
another potential mechanism of cross talk between the various PKC isoenzymes
(37). Therefore, it is
conceivable that the loss of signals generated by endogenous PKC
and
PKC
might have contributed in some way to the observed reduction in
SERCA2 mRNA. However, it should be pointed out that overexpression of
dnPKC
and dnPKC
increased, rather than decreased, SERCA2 mRNA
levels, suggesting that the effect of wtPKC
overexpression was due to a
direct effect of PKC
on the signaling pathways that regulate SERCA2
gene expression. In addition, overexpression of dnPKC
substantially
reduced endogenous PKC
but also increased (rather than decreased)
SERCA2 mRNA, suggesting that the effects of the PKC
adenoviruses were
also direct.
As demonstrated in Figs. 6
and 7, SERCA2 gene expression
under "basal" conditions appeared to be regulated by one or more
PKC isoenzymes, because selective inhibition of each PKC increased SERCA2 mRNA
levels in spontaneously contracting NRVM. dnPKC overexpression had by
far the greatest quantitative effect in upregulating SERCA2 mRNA. The present
results confirm our previous data, which showed that staurosporine and
chelerythrine increased SERCA2 mRNA levels in spontaneously contracting NRVM
(36). However, our previous
results need to be interpreted with caution, especially in light of new
information regarding the specificity and selectivity of these PKC inhibitors
in cultured cells. For instance, staurosporine, even at the low concentration
(10 nM) used in our previous study, inhibits many other signaling kinases
(15), and chelerythrine may in
fact have no inhibitory activity at all against PKC isoenzymes
(18). Both agents activated
the stress-activated protein kinases in NRVM
(26) and induced apoptosis
(26,
50). Indeed, these facts,
along with the potential for selective inhibition of specific PKC isoenzymes,
led to our use of the dnPKC adenoviruses in this system. However, it should be
pointed out that dnPKC
and dnPKC
overexpression also reduced
expression levels of endogenous PKC
and PKC
, respectively. These
somewhat surprising results may explain why dnPKC
significantly
increased basal SERCA2 mRNA levels, i.e., via an indirect effect on endogenous
PKC
. Future studies, including the use of isoenzymeselective,
cell-permeant peptide inhibitors of PKC translocation, may help to clarify
these issues (11).
In addition to the dnPKC mutants, we previously showed that inhibition of
contractile activity (by blockade of Ca2+ influx through
voltage-gated, L-type Ca2+ channels) also upregulated
SERCA2 mRNA levels (4). We also
found that a substantial proportion of endogenous PKC was found in the
membrane fraction of quiescent NRVM
(43), suggesting that this PKC
isoenzyme is substantially activated even under basal conditions. Electrical
stimulation of contraction caused the rapid translocation of additional
PKC
, along with PKC
, from the cytoplasm to a Triton X-100-soluble
membrane fraction (43). These
data suggest that the regulation of SERCA2 gene expression by
[Ca2+]i transients and contractile activity
may also be dependent on activation of one or both of the novel PKC
isoenzymes.
Although we provide evidence that PKC and PKC
are both
sufficient to downregulate SERCA2 mRNA, our data indicate that neither PKC
alone is necessary for this effect if the other novel PKC can be activated. A
similar redundancy in function has been demonstrated recently by Chen et al.
(12) in transgenic mice
overexpressing activator peptides of PKC
and PKC
in ventricular
myocytes. Both transgenic lines demonstrated similar degrees of cardiomyocyte
hypertrophy and upregulation of
-myosin heavy chain and atrial
natriuretic factor gene expression. However, the two lines differed
dramatically with respect to their susceptibility to ischemic injury,
indicating that PKC
and PKC
share common as well as distinct
signaling functions in the heart.
In this regard, it is interesting to speculate on what signaling pathways
may be operative downstream of PKC and PKC
and how they may
affect SERCA2 gene expression. First, there is evidence to indicate that
SERCA2 is regulated at both the transcriptional
(1,
2,
24,
45,
47) and posttranscriptional
(29,
36) levels, so it is
conceivable that the novel PKCs differentially influence SERCA2 mRNA levels by
affecting the rate of transcription vs. posttranscriptional processing and
stability of the SERCA2 mRNA. Second, there is also some evidence to suggest
that PKC
and PKC
may differentially influence SERCA2 gene
expression by activation of different MAPK cascades. Ho et al.
(27) provide convincing
evidence that SERCA2 expression in NRVM is regulated by the Ras-Raf-MEK-ERK
cascade. The same group has recently indicated that the
MKK6-p38MAPK pathway also reduces the activity of the rat SERCA2
promoter (1), leading to
reduced SERCA2 mRNA and protein levels and slowed relaxation of the
[Ca2+]i transient. Our laboratory has
recently shown that adenovirally mediated overexpression of PKC
predominantly activated ERKs, whereas PKC
overexpression predominantly
activated JNKs and p38MAPK under culture conditions identical to
those used in the present experiments
(26). These data are
consistent with dual regulation of SERCA2 gene expression by different MAPK
cascades at either the transcriptional or posttranscriptional level
(36). Future experiments are
necessary to identify the specific targets of novel PKC phosphorylation and to
determine exactly how the SERCA2 gene is regulated during hypertrophy and
HF.
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ACKNOWLEDGMENTS |
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These studies were supported by National Heart, Lung, and Blood Institute Grants R01-HL-34328 and R01-HL-63711 and by a gift to the Cardiovascular Institute from the Ralph and Marian Falk Trust for Medical Research. M. C. Heidkamp was the recipient of an National Institutes of Health National Research Service Award (F3-HL-68476), and Dr. Martin was the recipient of a James Beck/Patrick Scanlon, M.D. Scientist Development Award during the time these studies were performed.
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FOOTNOTES |
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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.
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