Article |
Address correspondence to Jeffery D. Molkentin, Division of Molecular Cardiovascular Biology, Department of Pediatrics, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039. Fax: (513) 636-5958. E-mail: jeff.molkentin{at}chmcc.org
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Abstract |
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Key Words: cardiac; hypertrophic growth; protein kinase C; signal transduction; MAPK
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Introduction |
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The protein kinase C family of calcium and/or lipid-activated serine-threonine kinases function downstream of nearly all membrane-associated signal transduction pathways (Molkentin and Dorn, 2001). Approximately 12 different isozymes comprise the PKC family, which are broadly classified by their activation characteristics. The conventional PKC isozymes (PKC, ßI, ßII, and
) are calcium- and lipid-activated, whereas the novel isozymes (
,
,
, and
) and atypical isozymes (
,
, v, and µ) are calcium independent but activated by distinct lipids (for review see Dempsey et al., 2000). Once activated, PKC isozymes translocate to discrete subcellular sites through direct interactions with docking proteins termed receptors for activated C kinases (RACKs)* (Mochly-Rosen, 1995), which permit specific substrate recognition and subsequent signal transduction. Different PKC isozymes or broad groups associate with unique RACKs as an important mechanism for regulating differential target specificities in vivo (Mochly-Rosen, 1995).
A number of reports have associated PKC activation with either cardiac hypertrophy, heart failure, ischemic injury, or agonist stimulation. For example, hemodynamic pressure overload stimulation in rodents can promote efficient translocation of PKC, ß,
,
, and
(Gu and Bishop, 1994; Jalili et al., 1999; Takeishi et al., 1999; De Windt et al., 2000). In cultured cardiomyocytes, diverse agonist and stress stimuli are also potent stimulators of PKC isozyme translocation (Clerk et al., 1996; Goldberg et al., 1997; Gray et al., 1997; Sil et al., 1998; Rohde et al., 2000). Using pharmacologic inhibitors, PKC activation has been implicated in regulating molecular events associated with agonist-induced cardiomyocyte hypertrophy (Glembotski et al., 1993; Yamazaki et al., 1995; Schluter et al., 1997; Sil et al., 1998). Isozyme-specific peptide inhibitors have also been employed in cultured cardiomyocytes and in transgenic mice to afford greater specificity of PKC inhibition. Specifically, overexpression of a PKCß C2 domain peptide in cardiomyocytes blocked phorbol estermediated calcium channel activity (Zhang et al., 1997), while PKC
inhibitory peptide or activating peptide affected inotropy and ischemia-induced cellular injury (Johnson et al., 1996a; Gray et al., 1997; Dorn et al., 1999).
More recently, transgenic mice have been generated with altered PKC isozyme signaling in the heart. Overexpression of either wild-type or a constitutively active deletion mutant of PKCß in a mouse heart was reported to induce cardiomyopathy (Bowman et al., 1997; Wakasaki et al., 1997), but more recent investigation has suggested that lower levels of expression or adult onset PKCß activation benefits cardiac contractility and ischemic recovery (Tian et al., 1999; Huang et al., 2001). Three groups have also reported transgenic mice with altered PKC activity in the heart. Expression of a PKC
-activating peptide in the mouse heart was associated with a physiologic activation of PKC
and an increase in myocyte cell number, but not cellular hypertrophy (Mochly-Rosen et al., 2000). In contrast, overexpression of an activated mutant PKC
in the mouse heart was reported to induce significant cardiac hypertrophy (Takeishi et al., 2000), but such a result is likely dependent on the absolute levels of PKC
overexpression and activity (Pass et al., 2001). Although a number of studies have demonstrated associations between various PKC isozymes and cardiac hypertrophy, the necessary and sufficient functions of specific PKC isozymes in the heart have not been established.
To evaluate potential functional divergence amongst PKC isozymes in regulating cardiomyocyte hypertrophy, cultured neonatal cardiomyocytes were subjected to adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC, ßII,
,
, and
(
wild-type only). PKC
was the only isozyme tested that was capable of inducing a hypertrophic response characterized by enhanced sarcomeric organization, increased cell surface area, increased atrial natriuretic factor (ANF) expression, and increased [3H]-leucine incorporation. Similarly, dominant negative PKC
, but not dominant negative ßII,
, and
, suppressed agonist-induced cardiomyocyte hypertrophy. PKC
-dependent regulation of cardiomyocyte hypertrophy was shown to require extracellular signalregulated kinase1/2 (ERK1/2) activation, suggesting a downstream mechanism of action. Collectively, these results implicate PKC
as a critical regulator of cardiomyocyte hypertrophic growth.
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Results |
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The observation that PE induced PKC translocation by immunocytochemistry is in contrast to four previous reports that failed to identify significant PKC
translocation to the particulate fraction after either PE or endothelin-1 stimulation of cardiomyocytes (Clerk et al., 1994, 1996; Deng et al., 1998; Hayasaki-Kajiwara et al., 1999). However, our analysis of translocation was performed in previously hypertrophied cardiomyocytes (stimulated with 2% fetal bovine serum for 24 h), which act in a more physiologic manner compared with smaller atrophic myocytes. Indeed, PE stimulation did not promote translocation of PKC
in atrophic myocytes, but significant translocation was readily observed in previously hypertrophied myocytes (Fig. 2 C, last lane) (see Discussion).
PKC uniquely induces hypertrophic growth
Although a large number of studies have demonstrated activation of PKC isozymes in association with cardiac hypertrophic growth, evidence of direct causality has not been established. To this end, each PKC isozyme was overexpressed in neonatal cardiomyocytes by adenoviral infection to evaluate their ability to induce hypertrophic growth. In serum-free medium, neonatal cardiomyocytes infected with a control ß-galactosidaseexpressing adenovirus (Adßgal) showed an atrophic phenotype without significant ANF protein expression (Fig. 3 A). However, -adrenergic agonist (PE) stimulation invoked sarcomeric organization, an increase in cell size, and ANF protein expression (perinuclear) (Fig. 3 A). Interestingly, overexpression of PKCßII,
,
, and
did not stimulate hypertrophic growth or ANF expression after 48 h (Fig. 3 A). In contrast, overexpression of wild-type PKC
promoted significant sarcomeric organization, increased cell size, and increased ANF protein expression (Fig. 3 A). Quantitation of three independent experiments demonstrated significantly greater cell surface area, percentage of cells expressing ANF, and [3H]-leucine incorporation in AdPKC
, but not AdPKCßII-, AdPKC
-, AdPKC
-, or AdPKC
-infected neonatal cardiomyocytes (Fig. 3, BD) (P < 0.05). Although these results suggest that PKC
is a unique inducer of cardiomyocyte growth, it was also important to verify the integrity of each adenoviral-expressed isozyme. To this end, PKC-specific enzymatic assays were performed from AdPKC
-, AdPKCßII-, AdPKC
-, AdPKC
-, and AdPKC
-infected cardiomyocytes, which each demonstrated an
5-fold increase in kinase activity compared with no infection or Adßgal infection (P < 0.05) (Fig. 3 E). In addition, control and AdPKC-infected cardiomyocytes were also stimulated with PE or PMA for 30 min to evaluate induction of kinase activity. PMA induced a further
7-fold increase in kinase activity in AdPKC
and AdPKCßII infected cardiomyocytes, while AdPKC
and AdPKC
infected cells showed an
3-fold increase in kinase activity (P < 0.05) (Fig. 3 E). A similar profile, albeit less robust, was observed after PE stimulation (Fig. 3 E).
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PKC is uniquely required for cardiomyocyte hypertrophy
It was of interest to examine the requirement of PKC isozymespecific activities as necessary mediators of agonist-induced cardiomyocyte hypertrophic growth. Accordingly, adenoviral vectors expressing dominant negative mutants of PKC, ßII,
, and
were used in cardiomyocytes. Each dominant negative mutant encodes the full-length protein, but contains a single amino substitution in a critical ATP binding residue, rendering the kinase inactive (Ohba et al., 1998; Matsumoto et al., 2001). Previous work has demonstrated that such mutations in PKC generate effective dominant negative proteins (Li et al., 1996; Hong et al., 1999; Pass et al., 2001; Strait et al., 2001). As assessed by Western blotting from three independent experiments, adenoviral infection at an moi of 100 pfu/ml resulted in
79-fold higher levels of each dominant negative isozyme 48 h postinfection compared with the wild-type isozymes (Fig. 6, A and B).
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Discussion |
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Mochly-Rosen and colleagues also showed PKC localization to the nucleus and perinucleus, whereas PKC
was localized in a cross-striated pattern in neonatal cardiomyocytes (Johnson et al., 1996a; Dorn et al., 1999). Although we identified a similar pattern of PKC
localization and redistribution, the pattern of PKC
translocation was somewhat different. Here it was shown that PKC
normally resides in the perinuclear membrane in unstimulated neonatal cardiac myocytes, but stimulation with PMA promoted movement out of the perinuclear region and into the nucleus itself (Fig. 2). PKCßII was shown previously to redistribute to the perinuclear area and to filamentous structures at the periphery of the neonatal cardiomyocyte (Disatnik et al., 1994). We also observed redistribution to the perinuclear region and the periphery of the cell, but a significant concentrations of PKCßII was also observed at cell-to-cell contacts upon stimulation with PMA in neonatal cardiomyocytes (Fig. 2). Although all four isozyme-specific antibodies readily detected their respective isozyme when overexpressed without loss of specificity, endogenous PKCßII and
were not readily detected despite positive accounts in the literature, suggesting either low protein abundance, partial epitope masking, or that previous descriptions may have employed antibodies with different specificity.
That PKC, ßII,
, and
each demonstrate unique subcellular localizations and redistribution patterns upon activation suggests regulation by isozyme-specific docking complexes. Indeed, PKC
, ßII,
, and
have each been shown to contain unique interacting domains in their NH2 terminus that provide specificity for anchoring proteins (RACKs), which regulate the specificity of substrate phosphorylation (for review see Mackay and Mochly-Rosen, 2001). A similar paradigm of isozyme-specific actions was also observed in cultured rat pituitary cells in which overexpression of PKC
, ßII,
, and
revealed a unique function for PKC
in regulating prolactin secretion (Akita et al., 1994). Collectively, numerous studies suggest that overexpression approaches can be employed to dissect isozyme-specific functions of PKC factors given the high degree of fidelity by which docking and substrate recognition are controlled in vivo.
PKC uniquely induces neonatal cardiomyocyte hypertrophic growth
Data implicating PKC isozymes as a regulators of cardiomyocyte hypertrophy have largely been derived by association. Specifically, agonist-induced cardiomyocyte hypertrophy has been shown to activate a diverse array of intracellular signaling factors, including PKC (for review see Molkentin and Dorn, 2001). To date, direct evidence implicating a specific PKC isozyme as a dominant regulator of myocyte hypertrophic growth is lacking. In culture, permeabilized neonatal cardiomyocytes treated with a generalized PKC pseudo-substrate peptide demonstrated reduced [14C]phenylalanine incorporation, suggesting a necessary role of PKC signaling in general (Johnson et al., 1996b). More recently, adenoviral-mediated overexpression of a constitutively active mutant of PKC was reported to enhance hypertrophic marker gene expression and to increase cell length, but interestingly, overexpression did not increase cell surface area or protein-to-DNA ratio (Strait et al., 2001). In this report, PKC
, but not PKCßII,
,
, or
, induced cardiomyocyte hypertrophic growth characterized by increased cell surface area, [3H]leucine incorporation and ANF expression.
It was of concern whether simple overexpression of a wild-type PKC isozyme would function similar to an activation event of that particular endogenous isozyme. Overexpression studies of other wild-type signaling factors have demonstrated efficacy through a net increase in both the activated and inactivated forms of the given factor, such that the inactivated state is innocuous while the activated state is functional. For example, overexpression of wild-type Gq or G
s in the mouse heart was sufficient to activate downstream targets (Iwase et al., 1996; D'Angelo et al., 1997). Consistent with these reports, overexpression of each wild-type PKC isozyme by adenoviral gene transfer produced a 57-fold increase in particulate association, suggesting greater activation by mass action.
Additional data examining PKC isozyme functions in regulating myocyte growth came from the use of transgenic mice expressing PKCß or PKC in the heart. Overexpression of wild-type or an activated deletion mutant of PKCß in the mouse heart was initially reported to induce a hypertrophic response (Bowman et al., 1997; Wakasaki et al., 1997). However, more recent investigation of PKCß transgenic mice suggests that physiologic activation of this isozyme in the adult heart does not promote cardiomyopathy and actually benefits cardiac contractility and ischemic recovery (Tian et al., 1999; Huang et al., 2001). Indeed, targeted disruption of the PKCß gene in the mouse did not affect the ability of these hearts to undergo hypertrophic growth (Roman et al., 2001). Similar disparity of interpretation surrounds the effect of PKC
in the hearts of transgenic mice. Expression of a PKC
activating peptide in the mouse heart, which promoted a physiologic activation of PKC
, did not result in cellular hypertrophy (Mochly-Rosen et al., 2000). In contrast, a second group reported that overexpression of an activated PKC
mutant protein in the mouse heart promoted cardiac hypertrophy (Takeishi et al., 2000). However, a third group demonstrated that high levels of PKC
overexpression promoted hypertrophy and cardiomyopathy, while more physiologic levels of expression were beneficial to the heart and rendered it resistant to myocardial ischemia (Pass et al., 2001). In general, these previous studies suggest a level of uncertainty as to whether PKCß or PKC
regulates the hypertrophic growth of cardiac myocytes.
PKC is required for neonatal myocyte growth
Overexpression of dominant negative mutants of PKC, ßII,
, and
in growth-stimulated neonatal cardiomyocytes suggested a necessary role for PKC
and PKC
. Such data implicate critical roles for these two latter isozymes as regulators of myocyte hypertrophy. However, AdPKC
dn infection promoted significant terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and poly(ADP-ribose) polymerase (PARP) protein cleavage, suggesting induction of apoptosis (unpublished data). Consistent with this interpretation, high levels of PKC
inhibitory peptide expression in the mouse heart by transgenesis induced a lethal cardiomyopathy, suggesting a critical role for basal signaling through PKC
in maintaining cellular homeostasis (Mochly-Rosen et al., 2000). Such results suggest that the inhibition of cardiomyocyte hypertrophic growth observed with AdPKC
dn is secondary to the loss of cell viability. In contrast, overexpression of dominant negative PKC
did not affect cardiomyocyte viability, suggesting that inhibition of hypertrophic growth is a direct mechanism and not due to the health of the cells. These results are also consistent with the unique ability of PKC
to induce myocyte hypertrophic growth.
The dominant negative PKC mutations consist of a lysine to arginine substitution in the ATP binding domain that render the kinases inactive but still able to interact with endogenous RACKs. Indeed, an identical PKC dominant negative mutation (K to R) was recently employed in cardiac-specific transgenic mice, which demonstrated competition with wild-type PKC
for RACK2 binding sites in vivo (Pass et al., 2001). Overexpression of dominant negative PKC
was also shown to only influence the translocation and activity of endogenous PKC
, and not other PKC isozymes (Strait et al., 2001). In other cell types, similar ATP binding site mutations produced potent and isozyme-specific dominant negative regulatory effects (Li et al., 1996; Hong et al., 1999; Pass et al., 2001; Strait et al., 2001). The above studies suggest that overexpression of kinase dead, dominant negative mutants of each PKC isozyme specifically antagonizes the activity of its cognate wild-type isozyme in vivo.
PKC regulates myocyte growth, in part, through ERK MAPK
The interconnectivity between PKC isozymes and MAPK signaling branches has been previously reported in many cell-types. In cardiac myocytes, dominant negative PKC was shown to down-regulate endothelin-1induced ERK1/2 activation (Strait et al., 2001), and in another study, agonist-induced ERK1/2 MAPK activation was correlated with PKC
activation (Jiang et al., 1996). In adult rabbit cardiomyocytes, adenoviral-mediated overexpression of PKC
was reported to activate ERK1/2 MAPK, yet a dominant negative PKC
mutant was reported to have no effect on basal ERK1/2 activation (Ping et al., 1999). More recently, PKC
and PKC
were also shown to be associated with ERK1/2 activation in cardiac myocytes, suggesting the involvement of other isozymes (Rohde et al., 2000).
In this report, we showed that overexpression of both PKC and PKC
induced significant ERK1/2 activation, but not p38 or JNK. These results are largely consistent with the reports discussed above. However, we also observed that dominant negative PKC
antagonized PMA-induced ERK1/2 activation, whereas dominant negative PKC
did not. At the functional level, dominant negative MEK1 (blocks ERK1/2 activation) inhibited wild-type PKC
-induced cardiomyocyte hypertrophy, suggesting a critical role for PKC
and ERK1/2 communication in regulating cardiomyocyte hypertrophic growth. Previous reports also support an interconnection between PKC
and ERK1/2 MAPK signaling, although the level at which PKC
interacts with the MAPK cascade is disputed (Kolch et al., 1993; Schonwasser et al., 1998).
Expression of dominant negative PKCßII, , and
did not significantly attenuate PMA-induced ERK1/2 activation in cardiomyocytes. PMA was employed since it acts as a more specific agonist of cPKC and nPKC activation, whereas hypertrophic agonists such as PE, endothelin-1, and angiotensin II stimulate diverse signaling pathways that could indirectly promote activation of selected PKC isozymes. Indeed, the studies discussed above, which demonstrated a role for PKC
and PKC
in regulating ERK1/2 activity, did not employ a direct PKC agonist such as PMA. In this manner, agonists that function through G-proteincoupled receptors or receptor tyrosine kinases likely utilize additional PKC isozymes, suggesting a complex relationship between PKC and ERK1/2 signaling pathways in cardiomyocytes.
The observation that dominant negative PKC attenuated PE-induced cardiomyocyte hypertrophy, coupled with the observation that PE augmented PKC
kinase activity and translocation to discrete intracellular locations by immunocytochemistry, suggests that G
q-coupled receptor signaling activates PKC
. This assertion is in contrast to four previous reports that failed to identify significant PKC
translocation to the particulate fraction after either PE or endothelin-1 stimulation of cardiomyocytes (Clerk et al., 1994, 1996; Deng et al., 1998; Hayasaki-Kajiwara et al., 1999). Two explanations may account for these differing results. First, it is largely assumed that PKC activation is synonymous with the ability to isolate PKC isozymes from a membrane-enriched or particulate protein fraction. This assumption may not be valid in every cell type or may vary depending on the stimulus and the specific isozyme analyzed. Second, our analysis of PE-induced PKC
translocation by immunocytochemistry and kinase activity was performed with hypertrophied neonatal cardiomyocytes (previously serum-stimulated), which are significantly larger and contain more organized sarcomeric structures. Cardiomyocytes in this condition may contain more organized docking units, which might be more amenable to detection of translocation. Alternatively, hypertrophied cardiomyocytes might have more complex membraneassociated signaling complexes, which could respond differently to PE stimulation. Indeed, atrophic cardiomyocytes were largely refractory to PE-induced PKC
translocation (Fig. 2 C). In any event, the conditions used here demonstrated PE-induced PKC
translocation and increased kinase activity in cultured cardiomyocytes that were in a more physiologic state (hypertrophied). Future analysis of PKC
signaling effects in animal models should shed additional light on the physiologic role of PKC
as a regulator of cardiac hypertrophy.
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Materials and methods |
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Replication-deficient adenovirus generation
The generation and characterization of adenovirus-encoding wild-type or dominant negative mutants of PKC, ßII,
,
, and
were described previously (Ohba et al., 1998; Matsumoto et al., 2001) (gift from Dr. Motoi Ohba, Tokyo, Japan). The dominant negative PKC
, ßII,
, and
cDNAs consisted of a lysine to arginine mutation in the ATP binding domain at amino acid positions 368, 371, 376, and 436, respectively. Each recombinant adenovirus was plaque purified, expanded, and titered in HEK293 cells using the agarose gel overlay method (Mittereder et al., 1996). Typical experiments involved infection of neonatal rat cardiomyocytes at a moi of 100 pfu for 2 h at 37°C in a humidified, 6% CO2 incubator. Subsequently, the cells were cultured in serum-free M199 media for an additional 24 h before treatments or analysis. Under these conditions
95% of the cells showed expression of the recombinant protein. Selected cultures were infected at a lower moi of 25 pfu/ml for immunocytochemical analysis so that nonexpressing cells could also be observed and compared with adenoviral infected cells.
Immunocytochemistry
Cardiomyocytes were prepared for immunocytochemistry as described previously (Taigen et al., 2000). To assess sarcomeric organization and cardiomyocyte hypertrophy antibody against -actinin (EA-53; Sigma-Aldrich) and ANF (Peninsula Laboratories) were used at a dilution of 1:500. Secondary antibodies included antimouse TRITC-conjugated antibody (Sigma-Aldrich) and antirabbit FITC-conjugated antibody (Sigma-Aldrich) at a dilution of 1:400. Quantitation of cardiomyocyte cell surface area was performed on
-actininstained cardiomyocytes using confocal laser microscopy and NIH Image software on a Sun system workstation. Characterization of PKC
, ßII,
, and
isozyme distribution also used confocal microscopy in conjunction with polyclonal antiserum purchased from Santa Cruz Biotechnology, Inc. (each used at 1:400).
-Tubulin antiserum (1:400) was purchased from Sigma-Aldrich.
Western blot analysis
Protein extracts were generated from cultured cardiomyocytes as described previously (Taigen et al., 2000). Protein samples were subjected to SDS-PAGE (8% gels), transferred to Hybond-P membrane (Amersham Pharmacia Biotech), blocked in 7% milk, and incubated with primary antibodies overnight in 3% milk at 4°C. Secondary antibodies IgG (alkaline phosphataseconjugated antimouse, rabbit, or goat) were incubated for 1 h at room temperature in 3% milk (Santa Cruz Biotechnology, Inc.). Chemifluorescent detection was directly performed with the Vistra ECF reagent (RPN 5785; Amersham Pharmacia Biotech) and scanned using a Storm 860 (Molecular Dynamics). Other antibodies included phospho-p38, p38, phospho-JNK, JNK wild-type, phospho-ERK1/2, and ERK1/2 (Cell Signaling).
PKC translocation assay tissue preparation and enzymatic assay
Neonatal cardiomyocyte cultures were prepared in homogenization buffer (25 mM Tris-Cl, pH 7.5, 4 mM EGTA, 2 mM EDTA, 5 mM DTT, 1 mM PMSF, and 1 ug/ml leupeptin) on ice and subsequently spun at 100,000 g for 30 min at 4°C. The supernatant product was saved as the cytosolic fraction while the remaining pellet was resuspended in homogenization buffer with the addition of 1% Triton X-100. The sample was then rehomogenized and incubated on ice for 30 min and spun again at 100,000 g for 30 min at 4°C, and the remaining supernatant fraction was saved as the particulate sample.
Total PKC activity was determined using a radioactive enzymatic assay (SignaTECT PKC assay system; Promega) in which cardiomyocyte supernatants are passed over a DEAE cellulose column to purify PKC proteins. PKC activity assays were performed in the presence of phospholipids (phosphatidylserine) and a PKC-biotinylated peptide substrate. All reactions were incubated at 30°C for 5 min, and [32P] incorporation was measured by transferring the completed reactions onto membranes (Promega).
Protein synthesis measurements
Rates of protein synthesis in cultured cardiomyocytes were determined by [3H]leucine incorporation. Cardiomyocytes were infected with adenovirus overnight, preincubated with leucine-free RPMI medium for 1 h, and then incubated with 2.5 µCi/ml [3H]leucine for 6 h. PE (20 µM) or vehicle was added to the cultures together with leucine-free medium and [3H]leucine incorporation was then quantified as described previously (Sadoshima et al., 1992).
Statistical analysis
Differences between data groups were evaluated for significance using a Student's t test of unpaired data or one-way analysis of variance and Bonferroni's post-test (± standard error of the mean).
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Footnotes |
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* Abbreviations used in this paper: ANF, atrial natriuretic factor; ERK1/2, extracellular signalregulated kinase1/2; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; moi, multiplicity of infection; PE, phenylephrine; pfu, plaque forming units; RACK, receptor of activated C kinases.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health and the Pew Charitable Trust Foundation (J.D. Molkentin) and by a National Institutes of Health training grant #5T32 HL07382 (J.C. Braz).
Submitted: 13 August 2001
Revised: 22 January 2002
Accepted: 28 January 2002
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