Reduction of angiotensin II-induced activation ofmitogen-activated protein kinase in cardiac hypertrophy
S. W. Rabkina,* P. S. Sungaa, J. S. Sangherab and S. L. Pelechb
aDepartment of Medicine, Biomedical Research Center, University of British Columbia, D 404, 2733 HeatherSt., Vancouver, B.C., V5Z 3J5 (Canada), Fax +1 604 875 5849, e-mail: [email protected] Pharmaceuticals, Vancouver, B.C. (Canada)
Received 3 June 1997; received after revision 7 October 1997; accepted 7 October 1997
predominantly p44ERK1 with lower amounts p42MAPK inAbstract. Mitogen-activated protein (MAP) kinasesrat ventricle, and no apparent changes with hypertro-play a role in cell growth and are activated in the heart
by cardiac stretch and various growth factors, but their phy. In hypertrophied hearts, ang II-induced MAP ki-role in signal transduction pathways once the heart has nase activity was less markedly increased and moreundergone hypertrophy is uncertain. To investigate the rapdily fell to baseline levels in comparison to theregulation of MAP kinases in the heart in response to response in nonhypertrophied hearts. Prolonged ang II
exposure did not produce the same effect on MAPangiotensin II (ang II), once cardiac hypertrophy haskinase activity in ventricles from Dahl S rats on abecome established, ventricular and skeletal muscle ex-
plants were studied from Dahl S salt-sensitive and Dahl low-salt diet, or skeletal muscle from salt-fed Dahl RR salt-resistant rats that were on a high (6% NaCl) salt and S rats. The ability of phorbol myristate acetate tosupplement in their diet. Cardiac hypertrophy was pro- simulate MAP kinase and ang II to simulate trans-duced in the Dahl S but not R rat through NaCl- location of protein kinase C from the cytosole to theinduced hypertension. MAP kinases were assayed by membrane was similarly compromised in hypertrophiedmyelin protein phosphotransferase activity in MonoQ ventricles. These results are consistent with a distur-
bance in the regulation of cell-signalling pathways infractions of cell extracts. Ang II increased MAP kinasescardiac hypertrophy in which the MAP kinase responsemainly in extracts from nonhypertrophic ventricles ofto ang II is dramatically altered.Dahl R rats on a high-salt diet. Immunoblots revealed
Key words. Mitogen-activated protein (MAP) kinase; cardiac hypertrophy; Dahl rat; angiotensin II.
Mitogen-activated protein (MAP) kinases function askey molecules in signalling processes stimulated bygrowth factors and in cell growth [1–4]. MAP kinasesare subject to regulatory control by various processesincluding activation by phosphorylation on tyrosineand threonine residues through stimulation of MAPkinase activators which are also protein kinases [1, 2, 5,
6]. MAP kinase kinase is activated by various upstreamkinases such as p74raf1 [1, 5–7]. The function(s) ofMAP kinases in the heart have not been well character-ized. Cardiac hypertrophy is analogous to cell growth invarious other cell types, as cardiomyocytes do not di-vide but respond to growth factors by increases in cellsize or hypertrophy. A role for MAP kinases in cardiachypertrophy is suggested by MAP kinase activation bycardiac stretch [8, 9], a potent stimulant of hypertrophiccardiac cell growth. MAP kinases can be activated by* Corresponding author.
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stimulation of cardiac cell surface receptors such asendothelin, fibroblast growth factors and a1-adrenergicreceptors [10–12] that also induce cardiac hypertrophy.Inhibition of MAP kinases prevent a1-adrenergic recep-tor-induced gene expression in cardiomyocytes [11].The octapeptide angiotensin II (ang II) exerts a spec-trum of effects on the cardiovascular system that mayinclude cardiac hypertrophy [13, 14]. A role for ang IIin myocardial hypertrophy is supported by the data thatang II stimulates cardiac protein synthesis [14, 15]. AngII activates MAP kinases in neontal rat cardiac cells [16,17] and in vascular smooth muscle cells [18, 19]. Al-though MAP kinases are implicated in the process ofcardiac hypertrophy, this is based on the study ofnormal cardiac cells. Inferences concerning the role ofagonist signal transduction pathways from normal (car-diovascular) cells may be at variance with their actionsin pathologic conditions where the pathways may nolonger be relevant. Some agonists that stimulate cyclicadenosine 3%,5%-monophosphate (cAMP)-dependentpathways can produce cardiac hypertrophy after acuteexposure and induce downregulation of these samepathways once hypertrophy is established [20–22]. Wehave demonstrated that the effect of ang II to modulatecAMP signalling is markedly perturbed in hyperten-sion-induced cardiac hypertrophy in the salt-sensitiveDahl S rat model of cardiac hypertrophy when com-pared with the genetically otherwise similar salt-resis-tant Dahl R rat that develops minimal cardiachypertrophy on a high-salt diet [22, 23]. Cardiac hyper-trophy is an important biological problem, as it is oneof the most powerful indicators of morbidity and mor-tality in patients with hypertension [24]. We sought totest the hypothesis that the regulatory control of MAPkinase activation by ang II is altered by the process ofcardiac hypertrophy.
Methods
Dahl rats. Sixteen inbred Dahl SR/Jr and 16 SR/Jr ratsfrom Harlan Sprague Dawley (Indianapolis, IN, USA)were maintained on a diet of powdered Purina Lab-chow supplemented with an additional 6% NaCl for 9to 10 weeks from weaning. The protocol was approvedby the University Committee on animal care. Animalswere permitted to drink tap water ad libitum. Thedegree of ventricular hypertrophy was assessed by de-termining the ventricle to total body weight ratio(mean91 SEM), which was 2.990.2 g/kg for 6%NaCl-fed Dahl R rats and 4.390.4 g/kg for 6% NaCl-fed Dahl S rats. Two inbred Dahl SR/Jr and two inbredSR/Jr rats were maintained on the same diet withoutadditional 6% NaCl and studied 9–10 weeks fromweaning.
Stimulation of hearts. Rats were anaesthetized using2.5% Halothane before sacrificing by cervical disloca-tion. Hearts were immediately removed and placed inoxygenated, 37 °C Dulbecco’s Minimal EssentialMedium (DMEM). Atria, major vessels and visibleconnective tissue were removed from the ventricles, theventricles weighed before being minced into cubes ap-proximately 1 mm3 and the pieces rinsed 2× in mediumto remove blood. In some experiments up to threehearts from identical strain, sex and ages were mixedtogether. The pieces were divided up and incubated with1 mM ang II or 100 nM phorbol myristate acetate(PMA) for 2 to 60 min at 37 °C in the presence of 95%O2 and 5% CO2.Preparation of ventricular extract. After the period ofstimulation, DMEM was aspirated from ventricularpieces. The pieces were rinsed with ice-cold homoge-nization buffer A (125 mM b-glycerol phosphate, pH7.2, 12 mM EDTA, 5 mM EGTA, 2 mM sodium ortho-vanadate, 1 mM dithiothreitol and 1 mM phenylmethyl-sulphonyl fluoride) and then placed in 1 ml of buffer A.Samples were homogenized using a microtip-equippedPolytron (Brinkman Instruments) at setting 7 for threebursts of 10 s per burst, in an ice bath. The ho-mogenates were centrifuged at 200,000g for 15 min in aBeckman TL-100 centrifuge, and the resultant superna-tant fractions (cytosol) were either processed immedi-ately or first stored frozen at −70 °C. The pellets werere-homogenized in 1 ml of buffer A that also contained1% Nonidet, and the detergent-solubilized extract wasobtained following centrifugation at 200,000g for15 min and also stored frozen at −70 °C.MonoQ chromatography. For MonoQ chromatogra-phy, �5 mg of cytosolic protein as loaded onto aMonoQ (1 ml) column equilibrated in column buffer B(25 mM b-glycerol phosphate, 10 mM MOPS, pH 7.2,5 mM EGTA, 2 mM MgCl, 1 mM dithiothreitol and2 mM sodium orthovanadate), at a flow rate of 1 ml/min. The column was developed with a 10-ml linear0–0.8 M NaCl gradient in buffer B at 1 ml/min using aPharmacia fast protein liquid chromatography system,and 250-ml fractions were collected.Kinase and protein assays. All kinase assays were per-formed after MonoQ fractionation of the ventricularextracts. MAP kinase activity with myelin basic protein(MBP) as a substrate was assayed as described previ-ously [25]. Protein kinase C (PKC) activity towardhistone H1 (Sigma type III-S) was measured as reported[26]. Using bovine serum albumin (BSA) as a standard,the protein concentrations of the extracts were deter-mined by the method of Bradford [27].Electrophoresis. SDS-polyacrylamide gel electrophore-sis (PAGE) was performed on 1.5-mm thick gels usingthe buffer system described by Laemmli [28]. An 11%separating gel and a 4% stacking gel were used. Sampleswere boiled for 5 min in the presence of 5× concen-
trated SDS-sample buffer (125 mM tris-HCl, pH 6.8,4% SDS, 0.01% bromophenol blue, 10% mercapto-ethanol and 20% glycerol) and electrophoresed for 17 hat 10 mA.Immunoblotting. Affinity-purified rabbit polyclonal an-tibody developed against a synthetic peptide patternedafter the C-terminus of rat extracellular regulatedprotein kinase 1 (ERK1) (ERK1-CT, CGG-P F T F D-M E L D D L P K E R L K E L I F Q U E T A R F O Q P G -APEAP). Rabbit polyconal anti-p44MARK antibodies,affinity-purified on a p44MARK-agarose column wereprepared as described [26]. Column chromatographyfractions of ventricular cytosol were subjected to SDS-PAGE. Subsequently, the separating gel was soaked intransfer buffer (25 mM tris, 192 mM glycine, 20%methanol) for 5 min and then sandwiched with a nitro-cellulose membrane. Proteins were transferred for 3 h at300 mA. Subsequently, the nitrocellulose membranewas blocked with TBS (tris-buffered saline) containing5% skim milk for 2 h at room temperature. The mem-brane was washed twice with TBS containing 0.05%Tween 20 (TTBS) for 5 min before incubation withrabbit polyclonal antibodies for MAP kinase (in 1%skim milk-TTBS; 1:1000 dilution) overnight at roomtemperature. The next day, the membrane was washedtwice with TTBS before incubation with the secondantibody [goat anti-rabbit immunoglobulin G (IgG) orgoat anti-mouse IgG coupled to alkaline phosphatase in1% skim milk-TTBS or 1% BSA-TTBS, respectively;1:3000 dilution] for 2 h at room temperature. Themembrane was rinsed with two washes of TTBS, fol-lowed by one wash with TBS before incubation with5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetra-zolium (Sigma) colour development solution (mixtureof 3% nitro blue tetrazolium in 1 ml of 70% dimethyl-formamide and 1.5% 5-bromo-4-chloro-3-indolyl phos-phate in 1 ml of 100% dimethylformamide beforeadding to 100 mg of 0.1 M NaHCO3, 10 mM MgCl2,pH 9.8). The colour was developed in 5 min to 4 h, andthe reaction was stopped by rinsing the membrane in alarge volume of water.
Results
Ang II stimulated MBP kinase activity within 5 mintreatment of ventricular explants from the Dahl R rats.This activity was confirmed to be attributed to MAPkinases, since the peak of MBP phosphorylation waseluted from MonoQ columns (fraction numbers 29–31),with 0.3 M NaCl (fig. 1A), a salt concentration fromwhich the MAP kinase isoforms p42MAPK and p44ERK1
have previously been shown to be consistently releasedfrom this resin [25, 26]. Further evidence that the angII-stimulated MBP phosphototransferase activity peakcorresponded to MAP kinases was also provided by
immunoblotting analysis of the MonoQ fractions withMAP kinase antibodies. The ERK1-CT antibodystrongly reacted with proteins of 42 and 44 kDa thateluted in fraction numbers 29–31 from the MonoQcolumns. The 42- and 44-kDa proteins were also visual-ized, to a lesser extent, with another antibody raisedagainst the purified sea star MAP kinase p44MAPK. Incontrast, in ventricular explants from the Dahl S ani-mal, on a high-salt diet, MBP kinase activity was mini-mally increased by ang II. The reduction in MAP kinaseactivity in ventricular explants from hypertrophic heartsof Dahl S rats (fig. 1B) was not due to decreases in theamount of p42MAPK and p44ERK1, which were compara-ble in Dahl R and Dahl S rat ventricular explants (fig.1C). The ERK1 band appears as a ‘tight’ band withslower mobility in the Dahl R rat samples, whereas inthe Dahl S, it is a broad band with increased mobility.This indicates that in the Dahl R rats, the majority ofthe ERK1 was in phosphorylated form (indicated bythe slower mobility) and activated form, whereas in theDahl S rats, there was the presence of both phosphoryl-ated and nonphosphorylated (inactive) forms.Ang II produced an initial increase in MAP kinaseactivity that declined with prolonged ang II treatment,and after 1 h MAP kinase activity was slightly belowcontrol (untreated) levels (fig. 2). The concentration of 1mM ang II was determined to be the optimal dose basedon preliminary ang II concentration-response data forMAP kinase activation. In ventricular explants fromhypertrophied hearts from the Dahl S rats, on high-saltdiets, MBP kinase activity was less than in Dahl R ratson high-salt diets. In hearts from Dahl S rats, ang II for5 min only partly stimulated MAP kinase activity, as itwas less than one-half the activity compared with angII-treated Dahl R rat hearts.When skeletal muscle explants from high-salt-fed DahlS and R rats were incubated with ang II for 20 min,MBP kinase activation was observed (fig. 3). In contrastto cardiac muscle, there was no blunting of the MBPkinase response in the Dahl S compared with the DahlR rat. Interestingly, there appeared to be a greaterincrease in MBP phosphorylation on eluates 29–31 inDahl S compared with Dahl R rats.To determine whether the MAP kinase response wasspecific for ang II, ventricular explants from high-salt-fed animals were treated with phorbol ester, PMA (fig.4). PMA, 100 nM, which directly activates PKC, pro-duced an activation of MAP kinase in Dahl R ventricu-lar explants comparable to what was achieved with angII. Like ang II, PMA did not activate MAP kinaseactivation in the Dahl S ventricular explants. Indeed, adecrease in MAP kinase activity was observed.To determine whether the effect of ang II was depen-dent on high-salt diet, Dahl S and R rats were kept onthe same diet without supplementation with NaCl. TheMAP kinase response to ang II was seen in ventricularexplants of Dahl S and R rats (fig. 5), and the threefold
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Figure 1. (A) MonoQ chromatography of extracts from high-salt-fed Dahl R and (B) results from Dahl S (right) ventricle explants.Cytosolic protein (5 mg) from control explants (0) and those treated with 1 mM ang II for 5 or 30 min. Column fractions were assayedfor phosphotransferase activity toward 1 mg MBP/ml. (C) Immunoblotting analysis of high-salt-fed Dahl R (left) and Dahl S (right)rat ventricular extracts with MAP kinase antibody. MonoQ column fractions from ang II-treated explants (A, B) were subjected toWestrn blotting analysis with anti-ERK1-CT peptide antibodies. Panel C is from samples electrophoresed on the same slab. Theelectrophoretic migrations of the prestained marker proteins (STD) ovalbumin (50 K) and carbonic anhydrase (33 K) are shown. Thedata are from a pair of Dahl S and R rats with the S rat shown 140% the weight of the R rat heart. Similar results were obtained inthree separate experiments.
response to ang II in Dahl S rats was similar to that inDahl R rats. The lower response in Dahl S rats may bedue to the mild cardiac hypertrophy in Dahl S rats evenon a low-salt diet [22]. To determine whether the MBPkinase response was specific to ang II, ventricular ex-plants, from high-salt-fed animals, were treated withang II, and PKC activity was measured in cytosolic andmembrane fractions. In ventricular extracts from DahlR rats, ang II, for 20 min, produced a partial conver-sion of PKC from its cytosolic form to a membrane-bound form (fig. 6), which is thought to be indicative ofactivation of this kinase. In contrast, similar ang IItreatment of ventricular explants from hypertrophiedhearts from high-salt-fed Dahl S rats did not activatePKC and appeared to have the opposite effect on thesubcellular distribution of PKC, i.e. less PKC was mem-
brane-associated and more was cytosolic (fig. 7). Therewas a significant (PB0.05, t test) difference between Rand S rats in the change in membrane PKC activity inresponse to ang II.
Discussion
Ang II-induced activation of MAP kinase was demon-strated in ventricular extracts from adult rat heart.Although ventricular explants contain both myocyteand fibrocyte elements, each has been shown in cultureto respond to ang II with an increase in MAP kinaseactivity in neonatal rat heart [16, 17]. In the Dahl ratventricle at least two MAP kinases of 42 and 44 kDawere evident with MAP kinase antibodies. The 42-kDa
Figure 2. The time course of MAP kinase activation is shown byMonoQ chromatography assayed for phosphotransferase activityin extracts, treated with 1 mM ang II, from rat ventricle fromanother pair of high-salt-fed Dahl R and Dahl S rats.
Figure 4. MBP phosphotransferase activities in the peak MonoQfractions corresponding to the p42 and p44 MAP kinases weredetermined as described in the legend to figure 1. Ventricularexplants were treated with PMA for 20 min (n=3) or untreated(n=3). The data are presented as the mean91 SEM.
latter isoform was the predominate MAP kinase in theventricular extracts, since the two MAP kinase antibod-ies employed in this study display similar degrees ofimmunoreactivity to these kinases. In other systemssuch as human A431 cells [25] and rat B lymphocytes[29], these same antibodies revealed higher levels of
species can be almost unequivocally identified asp42MAPK or ERK2 based upon its size [26]. The doubletof �44 kDa most likely corresponded to the phospho-rylated and dephosphorylated forms of p44ERK1. The
Figure 3. The MAP kinase response in skeletal muscle to ang II is shown in high-salt-fed Dahl R (A) and Dahl S (B). The MonoQchromatography of skeletal muscle extracts from high-salt-fed Dahl R and Dahl S rats. Cytosolic protein (5 mg) from control explants(0) and those treated with 1 mM ang II for 20 min were loaded on the columns.
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Figure 5. The MAP kinase response in ventricular muscle to ang II is shown in a pair of low-salt-fed Dahl R (A) and Dahl S (B).Cytosolic protein (5 mg) from control explants (0) and those treated with 1 mM ang II for 20 min were subjected to MonoQchromatography.
p42MAPK than p44ERK1. Ang II-induced activation ofMAP kinase in the ventricular myocardium of the DahlR was primarily directed toward p44ERK1. A proteinkinase C-dependent pathway may be responsible forp44erk1 activation in response to ang II, since PMA,which is a potent activator of protein kinase C (PKC),produced a degree of MAP kinase activation that wascomparable to that produced in response to ang II.PKC-dependent pathways are triggered by ang II-in-duced activation of a specific phospholipase C-catalysedhydrolysis of phosphatidylinositor-4,5-bisphosphate[30, 31]. Furthermore, ang II-induced PKC activationwas demonstrated in these hearts.In the hypertrophic ventricle, the degree and durationof ang II activation of p44ERK1 was reduced as com-pared with the nonhypertrophic rat ventricle. Specula-tion on a mechanism would include downregulation ofthe MAP kinase pathway in cardiac hypertrophy. Inyeast, the MAP kinase homologue, FUS3, downregu-lates its own phosphorylation pathway [32]. Thus MAPkinase activation produced by acute cardiac stretch orgrowth factors [8, 9] may have a negative feedback rolethrough MAP kinase-mediated phosphorylation of up-stream kinases. A more generalized effect on upstreamfactors is also suggested by the similar effect producedby phorbol ester and ang II in the Dahl S rat on ahigh-salt diet.Prolonged incubation with ang II appeared to be associ-ated with a faster disappearance of the MAP kinaseactivity in hypertrophied compared with nonhypertro-phied hearts. Since MAP kinase proteins were unaf-fected by hypertrophy, ang II-induced induction and
later reduction of MAP kinase activity in the Dahl Sheart did not reflect proteolysis of p44ERK1 or p42MAPK.Rather, a post-ang II receptor-binding step in the MAPkinase activation pathway may be compromised in themyocardium of Dahl S rats by a negative regulatorymechanism that is less apparent in Dahl R rats. Alter-natively, ang II-induced phosphatases [33] may be aug-mented in heart of Dahl S rats on high-salt diets. AMAP kinase phosphatase induced by growth factorsand phorbol ester reaches a maximum approximately30 min after stimulation [34], a time frame consistentwith increased inactivation of MAP kinase in Dahl Srats on high-salt diets. The altered ang II signal trans-duction that led to MAP kinase inhibition appeared tobe specifically linked to ventricular hypertrophy. It isnot present in the skeletal muscles or hearts of Dahl Srats fed low-salt diets. The limited ability of PMA tostimulate MAP kinase activation in the Dahl S rat heartimplies that PKC may be modified in the hypertrophicventricle such that altered PKC regulation in the hyper-trophic ventricular explants could account for the lossof ang II-induced MAP kinase activation. It has beenproposed that activation of PKC, when it occurs simul-taneously with activation of an src-like tyrosine kinasepathway, can antagonize activation of MAP kinasekinase [35].Although there are a number of different PKC iso-forms, we assessed the effect of ang II on calcium-sensi-tive PKC isoforms. Of the various kinds of PKCpresent in the heart, only the alpha- and beta-PKCisoforms are known to be calcium-sensitive [36]. Wehave found that the alpha-PKC isoform is the dominantone in rat cardiomyocytes (unpublished data).
Figure 6. Effect of ang II-induced translocation of protein kinaseC in ventricular explants treated with 1 mM ang II for 5 or 30 minand control explants (0). Samples containing 5 mg of protein fromcytosol and microsomal fractions were loaded on MonoQcolumns and each separately subjected to MonoQ chromatogra-phy. The Ca2+/diolein/phosphatidylserine-stimulated histone H1phosphotransferase activities of PKC in the monoQ fractions weredetermined. The data represent separate column fractionationsand set of kinase activity from a pair of Dahl S and R rats.Measurements were also made both in the absence and presenceof calcium, phospholipid and diacylglycerol.
rat has low plasma renin and ang II, which leads toenhanced response to ang II [37] and should, in princi-ple, lead to enhanced tissue response to in vitro ang IIstimulation.The blunted ang II-induced activation of MAP kinasemay be part of a generalized alteration of the signaltransduction pathways that initially produce cardiac cellgrowth. This hypothesis is supported by the findingsthat the MAP kinase response to both ang II and PKCwas similarly affected in cardiac hypertrophy. Cardiachypertrophy also displays dramatically altered regula-tory mechanisms involving cAMP-dependent signallingpathways [23]. Upstream abnormalities would includedifferent distribution of ang II receptor subtypes incardiac hypertrophy or postreceptor abnormalities in Gprotein composition with the development of cardiachypertrophy. Changes in ang II-R may occur in cardiachypertrophy [38] that may affect the interaction withvarious signal transduction pathways [39]. There are nodata on density and binding affinity of ang II receptorsor their subtypes in the Dahl rat, so conclusions aboutthese receptors must be made with caution. However,reduction in ang II number or affinity are not consistentwith the observation that ang II increases phosphoryla-tion of some cardiac proteins in cardiac hypertrophy inDahl S rats compared with Dahl R rats [40]. A moreattractive possibility is cardiac hypertrophy-induced Gprotein alterations. Bohm et al. [41] reported that DahlS rats on a high-salt diet had an increased immuno-detectable Gia, whereas the activity of Gsa -deficient S49cyc-mouse lymphoma cell membranes was unchanged.
The Dahl rat was selected for these studies because it isa model of genetic hypertension that develops cardiachypertrophy with an appropriate control. The Dahl S
Figure 7. The effect of ang II-induced translocation of protein kinase C in ventricular explants treated with 1 mM ang II for 20 minand control explants (0) is shown for Dahl R (left) and Dahl S (right) rats on a high-salt diet. The data are the mean91 SEM.
CMLS 53 (1997), Birkhauser Verlag, CH-4010 Basel/Switzerland958 Research Articles
Recent data have shown a link between cAMP and MAPkinase pathways [42]. We may speculate that an interac-tion between cAMP and mitogen-induced activation ofMAP kinases, in the heart, warrants investigation, sinceit might identify the underlying basis for ang II-alteredregulation of MAP kinase that we found in the presentstudy and the similar alteration in ang II effects oncAMP generation that we previously described in thismodel of cardiac hypertrophy [23].In summary, this study investigated MAP kinase activa-tion by ang II in heart that has already suffered hypertro-phy. Under these conditions, ang II produced less MAPkinase activation compared with the nonhypertrophicheart. Taken together with the marked alteration in thepattern of the MAP kinase response to PKC and thePKC response to ang II, this study points to profounddisturbances in intracellular regulation of signallingpathways involving MAP kinase in cardiac hypertrophy.
Acknowledgments. Supported in part by the Heart and StrokeFoundation of British Columbia & Yukon.
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