a farnesyltransferase inhibitor attenuates cardiac myocyte hypertrophy and gene expression

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J Mol Cell Cardiol 32, 1127–1140 (2000) doi:10.1006/jmcc.2000.1150, available online at http://www.idealibrary.com on A Farnesyltransferase Inhibitor Attenuates Cardiac Myocyte Hypertrophy and Gene Expression Angelino Calderone, Nadia Abdelaziz, Federico Colombo, Kathy L. Schreiber and Hansjo ¨rg Rindt Universite ´ de Montre ´al, Departe ´ment de Physiologie et Biochimie, et l’Institut de Cardiologie de Montre ´al, Montre ´al, Que ´bec, Canada (Received 6 August 1999, accepted in revised form 24 March 2000) A. C, N. A, F. C, K. L. S H. R. A Farnesyltransferase Inhibitor Attenuates Cardiac Myocyte Hypertrophy and Gene Expression. Journal of Molecular and Cellular Cardiology (2000) 32, 1127–1140. The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophic response, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present study utilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin- 1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with the farnesyltransferase inhibitor BMS-191563 (25 l) increased the level of unfarnesylated ras in the cytosolic fraction, and caused a concomitant 42±2% decrease in immunodetectable farnesylated ras in the particulate fraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3 H-leucine uptake (80±10% decrease; n=6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46±6% decrease; n=6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50±10% decrease (n=8; P<0.05) of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistent with the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulated extracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependent stimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, the pretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANP and -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression of the natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present study has shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate the hypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the small GTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathways are also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneity in their recruitment. 2000 Academic Press K W: Myocyte hypertrophy; Gene expression; Farnesyltransferase inhibitor; ras; Calmodulin; G-protein coupled receptors. myocardial function in response to a chronic hemo- Introduction dynamic load, or following myocardial infarction. At the cellular level, myocyte hypertrophy is char- Cardiac hypertrophy, defined as an increase in myo- cyte cell size without a change in cell number, acterized by an increase in protein and RNA syn- thesis, and the synthesis of new sarcomeres. 1,2 represents the primary mechanism for preserving Please address all correspondence to: Dr Angelino Calderone, Institut de Cardiologie, 5000 rue Belanger est, Montre ´al, Que ´bec, Canada H1T 1C8. Tel: 514-376-3330 ext 3710. Fax: 514-376-1355. E-mail: [email protected] This work was supported by the Medical Research Council of Canada, and the Heart & Stroke Foundations of Canada and Quebec. 0022–2828/00/061127+14 $35.00/0 2000 Academic Press

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Page 1: A Farnesyltransferase Inhibitor Attenuates Cardiac Myocyte Hypertrophy and Gene Expression

J Mol Cell Cardiol 32, 1127–1140 (2000)

doi:10.1006/jmcc.2000.1150, available online at http://www.idealibrary.com on

A Farnesyltransferase InhibitorAttenuates Cardiac Myocyte Hypertrophyand Gene ExpressionAngelino Calderone, Nadia Abdelaziz, Federico Colombo, Kathy L. Schreiberand Hansjorg RindtUniversite de Montreal, Departement de Physiologie et Biochimie, et l’Institut de Cardiologie deMontreal, Montreal, Quebec, Canada

(Received 6 August 1999, accepted in revised form 24 March 2000)

A. C, N. A, F. C, K. L. S H. R. A Farnesyltransferase Inhibitor AttenuatesCardiac Myocyte Hypertrophy and Gene Expression. Journal of Molecular and Cellular Cardiology (2000) 32,1127–1140. The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophicresponse, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present studyutilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin-1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with thefarnesyltransferase inhibitor BMS-191563 (25 l) increased the level of unfarnesylated ras in the cytosolicfraction, and caused a concomitant 42±2% decrease in immunodetectable farnesylated ras in the particulatefraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3H-leucine uptake (80±10%decrease; n=6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46±6%decrease; n=6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50±10% decrease (n=8; P<0.05)of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistentwith the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulatedextracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependentstimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, thepretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANPand -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression ofthe natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present studyhas shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate thehypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the smallGTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathwaysare also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneityin their recruitment. 2000 Academic Press

K W: Myocyte hypertrophy; Gene expression; Farnesyltransferase inhibitor; ras; Calmodulin;G-protein coupled receptors.

myocardial function in response to a chronic hemo-Introductiondynamic load, or following myocardial infarction.At the cellular level, myocyte hypertrophy is char-Cardiac hypertrophy, defined as an increase in myo-

cyte cell size without a change in cell number, acterized by an increase in protein and RNA syn-thesis, and the synthesis of new sarcomeres.1,2represents the primary mechanism for preserving

Please address all correspondence to: Dr Angelino Calderone, Institut de Cardiologie, 5000 rue Belanger est, Montreal, Quebec, CanadaH1T 1C8. Tel: 514-376-3330 ext 3710. Fax: 514-376-1355. E-mail: [email protected]

This work was supported by the Medical Research Council of Canada, and the Heart & Stroke Foundations of Canada and Quebec.

0022–2828/00/061127+14 $35.00/0 2000 Academic Press

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A. Calderone et al.1128

Associated with the hypertrophic response is the pertrophic stimuli remains to be clearly defined. Inthis regard, the present study employed a phar-transcriptional induction of immediate early genes

(e.g. c-fos, c-jun, and c-myc), and the recapitulation macological approach to acquire a better under-standing as to the role of ras and calmodulin inof cardiac fetal genes, (e.g. prepro-atrial natriuretic

peptide (prepro-ANP), b-myosin heavy chain, and myocyte hypertrophy in response to two putativehypertrophic stimuli, norepinephrine, and en-skeletal a-actin).3–6 Previous studies have dem-

onstrated the nature of the hemodynamic load dothelin-1. To assess the role of ras, cardiac myo-cytes were treated with the farnesyltransferaseimposed on the heart plays an important role in

determining the morphological and molecular inhibitor BMS-191563, a peptidomimetic com-pound which prevents ras farnesylation and itshypertrophic phenotype.5,7 Moreover, numerous

studies support the concept that the concomitant subsequent translocation to the plasma membrane,whereas the role of calmodulin was assessed withactivation of the sympathetic system, and the re-

cruitment of putative hypertrophic factors such the pharmacological inhibitor W7. Using this strat-egy, the present study has demonstrated no-as angiotensin II, endothelin-1, and transforming

growth factor-b may initiate and/or propagate the repinephrine and endothelin-1 stimulated myocytehypertrophy and natriuretic peptide mRNA ex-hypertrophic phenotype in response to a hemo-

dynamic load.8,9 pression was attenuated by the farnesyltransferaseinhibitor BMS-191563. Likewise, W7 attenuatedThe neonatal rat ventricular myocyte cell model

has been extensively used to identify a plethora of the hypertrophic response of norepinephrine,whereas endothelin-1 stimulated cardiac myocytehypertrophic stimuli, characterized by the ability

to promote cardiac myocyte growth, and induce a hypertrophy was unaffected. These data dem-onstrate the hypertrophic response of at least twopattern of gene expression reminiscent of in vivo

cardiac hypertrophy in the adult rat.6 Using the in physiologically relevant stimuli is sensitive to thenegative modulation by a farnesyltransferase in-vitro cell model, the small GTP-binding protein

ras has been identified as an important proximal hibitor, thereby supporting a potential role of ras.However, alternative ras-independent signalingsignaling event implicated in the progression of

hypertrophy.10–12 The direct injection of oncogenic pathways also contribute to the hypertrophicphenotype, and there appears to exist a stimulus-Hras into neonatal rat cardiac myocytes was shown

to increase cell size, reorganize contractile proteins, specific heterogeneity in their recruitment.and elicit the concomitant transcriptional ex-pression of an ANP-promoter construct.11,12 How-ever, at least one study demonstrated that Experimental Proceduresoncogenic ras was unable to promote the re-organization of contractile proteins into sarcomeric Cultured neonatal rat cardiac myocytesstructures, a hallmark feature of cardiac myocytehypertrophy.13 An alternative hypertrophic sig- Neonatal rat cardiac myocytes were isolated as

previously described.16 Briefly, hearts were removednaling pathway involves Ca2+, as elevated intra-cellular Ca2+ promotes growth, and the synthesis from 1–3-day-old Sprague-Dawley neonatal rats

(Charles River Canada; St Constant, Quebec) an-and release of ANP from cardiac myocytes.14–17 Acritical element of Ca2+-dependent pathways is the esthetized with ether and killed by decapitation.

Ventricular tissues were digested with 0.1% trypsincalcium binding protein calmodulin, which recruitsvarious effector molecules, including calmodulin (GIBCO BRL; Burlington, Ontario) in HBSS (Ca2+-

free; GIBCO BRL) overnight at 4°C. Ventricular cellskinase and calcineurin.17–19 Evidence of a hy-pertrophic role for calmodulin was demonstrated were then recovered by repeated digestion of the

tissue in 10 ml of 0.1% collagenase (GIBCO BRL)in transgenic mice as the overexpression of thisprotein was sufficient to induce cardiac hyper- in HBSS (Ca2+-free). The supernatants collected

from each digestion were centrifuged at 100 g fortrophy.20 In neonatal rat cardiac myocytes, theoverexpression of either calmodulin kinase or cal- 3 min at 4°C. The pellets were resuspended in ice

cold HBSS, pooled, and centrifuged at 100 g forcineurin promoted the expression of an ANP-pro-moter construct.18,19 3 min at 4°C, and subsequently resuspended in

DMEM containing 7% heat-inactivated FBS (GIBCOAlthough the overexpression of ras and cal-modulin in both in vitro and in vivo settings were BRL). Cells were then preplated twice on T75 flasks

for 60 min to enrich for myocytes and decreaseshown to be sufficient to promote cardiac myocytehypertrophy, the recruitment of these proximal sig- contamination of non-muscle cells. The non-ad-

herent cardiac myocytes were then plated at analing events by physiologically relevant hy-

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1129

density of 100–200 cells/mm2 in DMEM containing Measurement of inositol phosphate levels7% FBS for 24 h, and subsequently changed toserum-free DMEM containing insulin (5 lg/ml), Total inositol phosphate levels were measured as

previously described.22 Briefly, cardiac myocytestransferrin (5 lg/ml), and sodium selenite (5 ng/ml; Collaborative Biomedical; Bedford, MA, USA), were plated at a density of 100–200 cells/mm2 in

60-mm plates (Falcon), and incubated for a period ofand all experiments were performed 24 h later.Previous studies have shown norepinephrine-stim- 24 h with 1 lCi/ml of 3H-myo-inositol (Amersham

Pharmacia; Baie D’Urfee, Quebec). Cardiac myo-ulated neonatal rat cardiac myocyte hypertrophyoccurs exclusively via activation of the a1-ad- cytes were then incubated with 50 m LiCl for

15 min prior to the addition of either no-renergic receptor when cells were plated at lowdensity (100–200 cells/mm2).16,21 repinephrine (1 l) or endothelin-1 (10 n), and

agonist stimulation proceeded for an additional30 min. The total inositol phosphate pool (inositol-phosphate, -bisphosphate, and -trisphosphate) wasseparated by ion-exchange chromatography(AGX18 resin; Bio-Rad Laboratories Canada). TheDetermination of myocyte cell sizetotal inositol phosphate pool was expressed as aratio of 3H-myo-inositol incorporation into the phos-Cardiac myocytes were plated at a density of 100–

200 cells/mm2 and pretreated for 24 h with BMS- phatidylinositol lipid pool, as previously described.22

191563 (25 l; Bristol Myers Squibb, Princeton,NJ, USA; Dr V. Manne) prior to the addition ofnorepinephrine (1 l; Sigma; St Louis, MI, USA). Distribution of particulate and cytosolic rasFollowing a 24 h stimulation with norepinephrine,cardiac myocyte cell size was determined by Cardiac myocytes were plated at a density of 100–

200 cells/mm2 in 100 mm plates (Falcon) andplanimetry using a computer-assisted histo-morphometry and the NIH Image 1.6 imaging treated with either vehicle (DMEM) or BMS-191563

(25 l) for 24 h. Cells were subsequently washedsoftware. Cells were randomly chosen by a tech-nician unaware of the experimental protocol. Car- twice with PBS (pH 7.4; 4°C), and resuspended in

1 ml of a buffer containing 20 m HEPES (pH 7.5),diac myocyte length (lm) reflects the entireperimeter of the cell, and surface area is represented 20 m b-glycerophosphate, 20 m NaF, 0.2 m

Na3VO4, 5 m EDTA, 5 m EGTA, 0.5 m PMSF,as lm2.25 lg/ml leupeptin, and 5 m DTT. The homo-genate was centrifuged for 30 min at 100 000 g(5°C) to yield supernatant (cytosolic) and pelletfractions. The pellet was resuspended in buffer Aplus 1% (v/v) triton X-100, and centrifuged as3H-leucine uptake studies in cardiac myocytesbefore to yield a supernatant which contains theparticulate fraction.23 Samples from the cytosolicCardiac myocytes plated at a density of 100–200

cells/mm2 (24-well plates; Falcon) were pretreated and particulate fractions (50–100 lg) were resolvedin a 10% polyacrylamide-SDS gel, and transferredfor 24 h with the farnesyltransferase inhibitor BMS-

191563 (25 l), or 30 min with the MEK inhibitor onto a nitrocellulose membrane (Amersham Phar-macia). The membrane was probed with antiseraPD098059 (50 l; Biomol; Plymouth Meeting, PA,

USA), or 1 h with the calmodulin inhibitor W7 which recognizes the three ras isoforms H,K,N (panras (AB-3); 1:500; Calbiochem), and detected by(5 l; Calbiochem; Lajolla CA, USA), prior to the

addition of agonist. Subsequently, norepinephrine Enhanced Chemiluminescence (ECL, Dupont NEN).Quantification of the bands was performed with(1 l; Sigma; St Louis, MI, USA) or endothelin-1

(10 n; Calbiochem) was added for a period of a laser densitometer (Chemilmager 4000 I v4.04software; Alphan Innotech).24 h in the presence of 2 lCi/ml of 3H-leucine (ICN

Biomedicals Inc; Costa Mesa, CA, USA). Followingthe 24 h period of agonist treatment, cardiac myo-cytes were washed with PBS (pH 7.4; 4°C), and Extracellular-signal regulated kinase (ERK) assaycold 5% TCA was added for 30 min to precipitateprotein. The precipitates were washed twice with Cardiac myocytes plated at a density of 100–200

cells/mm2 (100 mm plates; Falcon) were pretreatedcold water and resuspended in 0.4 NaOH, andaliquots were counted in a scintillation counter. for 24 h with BMS-191563 (25 l) prior to a 5 min

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A. Calderone et al.1130

stimulation with either norepinephrine (1 l) or by size electrophoresis on a 1.3% agarose/4% form-aldehyde gel (15 lg total RNA/sample). RNA wasendothelin-1 (10 n). Following agonist stimu-

lation, cardiac myocytes were washed twice with transferred to nylon membranes (Genescreen plus;Dupont-NEN, Canada) by vacuum blotting (Bio-PBS (pH 7.4; 4°C), and lysed in a buffer containing

10 m TRIS (pH 7.5), 150 m NaCl, 1 m EDTA, Rad laboratories, Canada LTD, Mississauga, On-tario), and ultra violet cross-linked (Amersham1 m EGTA, 50 m NaF, 20 m b-gly-

cerophosphate, 0.5 m phenylmethylsulfonyl Pharmacia).A 0.7-kb fragment of rat prepro-atrial natriureticflouride, 1 m sodium vanadate, 1% triton X-100,

0.5% nonidet P-40, and 1 lg/ml of leupeptin and peptide (courtesy of Dr M. Boluyt), a 0.6-kb frag-ment of rat prepro-brain natriuretic peptide (cour-aprotinin. Immunoprecipitation of ERK was per-

formed by the addition of the anti-ERK-1 and -ERK- tesy of Dr M. Nemer), and a 1.2-kb fragment of ratGAPDH (American Type Culture Collection; Rock-2 goat polyclonal antibodies (1:150; Santa Cruz

Biotechnology; Santa Cruz, CA, USA) to 200 lg of ville, MD, USA) were labeled with a-32P-dCTP(Amersham Pharmacia) to a specific activity ofmyocyte lysate for 4 h at 4°C. The im-

munecomplexes were recovered by the addition of 1–2×106 cpm/ng cDNA by the random hexamerpriming method and hybridized to nylon mem-protein A-G agarose (Santa Cruz Biotechnology),

the pellets were washed twice with lysis buffer, and branes for 18–24 h at 42°C, as previously de-scribed.16 The filters exposed to the cDNA probestwice with kinase reaction buffer (50 m HEPES,

20 m MgCl2, pH 7.4). The ERK assay was per- were washed twice (15 min, room temperature)with 300 m NaCl/30 m trisodium citrate andformed in 50 ll volume of kinase reaction buffer

containing 2 lCi/ml of c-32P-ATP (Amersham Phar- 0.1% SDS and twice (15 min, 45°C) with 30 m

NaCl/3 m trisodium citrate and 0.1% SDS. Nylonmacia), 10 lol/l of unlabeled ATP, 0.2 mg/ml ofmyelin basic protein, at 30°C for 30 min. The re- membranes were subsequently exposed to Kodak

XAR film with an intensifying screen at −70°C,action was stopped by the addition of 20 ll of200 m of EDTA (pH 7), and a 20 ll aliquot of the and films were scanned with a laser densitometer

(Chemilmager 4000 I v4.04 software; Alphan In-reaction mixture was spotted onto Whatman P-81paper (VWR-Canlab; Mont-Royal, Quebec), washed notech). All levels of mRNA reported in this paper

are normalized to the level of GAPDH mRNA.4× with 75 m H3PO4, once with acetone, andsubsequently counted in a scintillation counter.Data are expressed as fold increase in radioactivityincorporated into myelin basic protein, as compared Statistical methodsto unstimulated cardiac myocytes. Alternatively,3× SDS-sample buffer was added to a 25 ll aliquot, Data are presented as the mean±SEM. Statisticalboiled, and subjected to SDS-polyacrylamide gel analysis was performed by Student’s unpaired t-test(10%) electrophoresis in order to visualize phos- (two-tailed), and a value of P<0.05 was consideredphorylated myelin basic protein by auto- significant.radiography.

ResultsAssessment of steady-state mRNA levels

The role of ras in norepinephrine- and endothelin-1-stimulated protein synthesis in cardiac myocytesCardiac myocytes plated at a density of 100–200

cells/mm2 in 100 mm plates (Falcon) were pre-To assess the efficacy of the peptidomimetic farne-treated with either BMS-191563 (25 l) or W7

(5 l) for 24 and 1 h, respectively, prior to the syltransferase inhibitor BMS-191563, the dis-tribution of particulate (membrane-bound) andadministration of either norepinephrine (1 l) or

endothelin-1 (10 n). Following a 24 h period of cytosolic ras in neonatal rat cardiac myocytes wasexamined. In untreated cardiac myocytes, ras wasagonist stimulation, total RNA was isolated by a

modification of the technique of Chomczynski and predominantly localized in the particulate fraction,consistent with the posttranscriptional farne-Sacchi.24 Cardiac myocytes were resuspended in

denaturing solution (4 M/L guanidium thiocyanate, sylation of the protein (Fig. 1). In the cytosolicfraction, a weak signal was detected indicating a25 m sodium citrate, 0.5% laurylsarcosine, 0.1

mercaptoethanol pH 7). Total RNA was denatured fraction of ras was not membrane-bound in thebasal state. Moreover, the slower electrophoreticwith formaldehyde and formamide, and separated

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1131

Figure 1 The inhibition of ras protein processing by the farnesyltansferase inhibitor BMS-191563. In untreated cardiacmyocytes (control), ras was predominantly detected in the particulate (PAR) fraction, consist with the post-transcriptionalprocessing (P; farnesylation) of the protein. By contrast, a weak signal was detected with a slower electrophoreticmobility in the cytosolic (CYT) fraction, highlighting the absence of post-transcriptional processing (U) of ras. A 24 htreatment of cardiac myocytes with BMS-191563 (BMS; 25 l) increased the level of unfarnesylated ras (U) in thecytosolic (CYT) faction, which coincided with a 42±2% decrease (n=2) in detectable farnesylated ras (P) in theparticulate (PAR) fraction.

mobility of this ras signal in the cytosolic fraction 3H-leucine uptake by 80±10% (P<0.01; n=6)(Fig. 4). In parallel experiments, the exposure (24 h)reflects the absence of farnesylation (Fig. 1). A 24 h

treatment with BMS-191563 (25 l) resulted in of cardiac myocytes to endothelin-1 (10 n) in-creased 3H-leucine uptake by 42±5% (P<0.01; n=an increase of unfarnesylated ras in the cytosolic

fraction, which coincided with a 42±2% decrease 6). Likewise, the pretreatment of cardiac myocyteswith 25 l BMS-191563 caused a significant de-(n=2) in immunodetectable farnesylated ras in the

particulate fraction. Thus, these data confirm BMS- crease (46±6%; n=6; P<0.05) of endothelin-1mediated 3H-leucine uptake, albeit less pronounced,191563 can inhibit the farnesylation of the small

GTP-binding protein ras in neonatal rat cardiac as compared to norepineprine (Fig. 4).myocytes.

In untreated neonatal rat cardiac myocytes, celllength and surface area were 33±2 lm (n=9), The role of calmodulin in norepinephrine- and

endothelin-1-stimulated protein synthesis in cardiacand 302±28 lm2 (n=9), respectively. The surfacearea of cardiac myocytes treated for 24 h with myocytes25 l BMS-191563 was similar (356±50 lm2;n=10) to the untreated cells, whereas a modest Previous studies have demonstrated a role of cal-

modulin-dependent pathways in cardiac myocytesignificant increase in cell length was observed inthe BMS-191563 treated cells (42±2 lm; n=10; hypertrophy, and both norepinephrine and en-

dothelin-1 can mobilize intracellular calcium inP<0.05 v untreated cells). The 24 h exposure tonorepinephrine (1 l) increased cardiac myocyte part via the synthesis of the second messenger

inositol 1,4,5-trisphosphate. Consistent with pre-cell size, and the pretreatment with BMS-191563attenuated the hypertrophic response (Figs 2 and vious studies, norepinephrine (1 l) stimulation

(30 min) of cardiac myocytes increased intracellular3). Consistent with these observations, the exposureof cardiac myocytes to norepinephrine (1 l) for inositol phosphate levels (0.122±0.01; P<0.001;

n=7), as compared to unstimulated myocytes24 h caused a 53±8% (P<0.01; n=6) increase in3H-leucine uptake (Fig. 4). A 48 h treatment with (0.052±0.01). Likewise, endothelin-1 (10 n)

stimulation promoted a >6-fold increase of intra-25 l BMS-191563 resulted in a modest decreasein basal 3H-leucine uptake (14±3% decrease; n= cellular inositol phosphate levels (0.299±0.03;

P<0.001; n=5), as compared to unstimulated myo-6). The 24 h pretreatment of cardiac myocytes withBMS-191563 prior to the addition of no- cytes. Thus, the IP3-mediated increase of intra-

cellular Ca2+ supports the potential recruitment ofrepinephrine attenuated the subsequent increase in

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A. Calderone et al.1132

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1133

Figure 3 The regulation of cardiac myocyte cell size by the farnesyltransferase inhibitor BMS-191563. The 24 htreatment with norepinephrine (NE; 1 l) caused a significant increase in cardiac myocyte surface area (n=9; P<0.01),and length (n=9; P<0.01), as compared to untreated cells. The pretreatment (24 h) with 25 l BMS-191563 attenuatedthe norepinephrine-mediated increase in cardiac myocyte surface area (n=10), and length (n=10). (∗) denotes P<0.05,and (∗∗) denotes P<0.01 v NE.

calmodulin by both agonists. A 24 h treatment stimulation of cardiac myocytes with norepinephrinewith the calmodulin inhibitor W7 (5 l) caused a (1 l) or endothelin-1 (10 n) increased ERK activitymodest decrease in basal 3H-leucine uptake by 4±1-fold, and 15±6-fold (n=3 for each; Fig. 5),(12±4% decrease; n=7). The pretreatment (1 h) respectively, as compared to unstimulated myocytes.of cardiac myocytes with W7 prior to the addition The pretreatment (24 h) with 25 l BMS-191563of norepinephrine attenuated the subsequent in- caused a 96±4% (P<0.01; n=3), and a 68±6%crease in 3H-leucine uptake by 50±10% (P<0.05; (P<0.01; n=3) decrease of norepinephrine- and en-n=8) (Fig. 4). By contrast, W7 pretreatment did dothelin-1-stimulated ERK activity, respectively (Fig.not alter endothelin-1 stimulated 3H-leucine uptake 5). Thus, these data demonstrate norepinephrine-(Fig. 4). and endothelin-1-mediated ERK activation occurred

via a ras-dependent pathway, and supports a potentialrole of ERK in the hypertrophic response. To examinethis latter premise, cardiac myocytes were treatedNorepinephrine- and ET-1-stimulated ERK activity iswith the PD098059, a putative pharmacologicalattenuated by the farnesyltransferase inhibitor BMS-agent which inhibits the upstream activator of ERK,191563; ERK activation is not necessary for thethe mitogen-activated protein kinase kinase (MEK).26hypertrophic growth responseThe pretreatment for a period of 30 min with theMEK inhibitor PD098059 (50 l) did not attenuateThe serine/threonine extracellular signal-regulatednorepinephrine- or endothelin-1-stimulated 3H-leu-kinase (ERK) represents a putative downstream target

of the small GTP-binding protein ras.25 A 5 min cine uptake (Fig. 5).

Figure 2 The effect of BMS-191563 on the norepinephrine-mediated increase of cardiac myocyte cell size. The 24 hexposure of cardiac myocytes to norepinephrine (1 l) caused an increase in cardiac myocyte cell size (panel B), ascompared to untreated cells (panel A). The 24 h treatment with 25 l BMS-191563 did not alter the cellular phenotypeof cardiac myocytes (panel C), as compared to untreated cells (panel A). However, the pretreatment with BMS-191563attenuated the norepinephrine-mediated increase in cardiac myocyte cell size (panel D), as compared to norepinephrine-treated cells (panel B). Cardiac myocytes were photographed at 100× magnification utilizing the Nikon Diaphot 200microscope.

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A. Calderone et al.1134

Figure 4 The regulation of protein synthesis in cardiac myocytes by the farnesyltransferase inhibitor BMS-191563and the calmodulin inhibitor W7. (A) The norepinephrine (NE; 1 l) and endothelin-1 (ET-1;10 n) mediated increaseof 3H-leucine uptake in cardiac myocytes was decreased by the pretreatment with the farnesyltransferase inhibitorBMS-191563 (25 l). (B) The pretreatment with W7 (5 l) attenuated NE-stimulated 3H-leucine uptake, whereas theET-1 response was unaffected. (∗) denotes P<0.05 v ET-1 or NE, and (∗∗) denotes P<0.01 v NE.

The role of ras and calmodulin in norepinephrine- and (10 n) caused a 3.6±0.6-fold (n=4) andendothelin-1-stimulated prepro-ANP and -BNP mRNA 4.7±0.9-fold (n=4) increase in the steady-stateexpression in cardiac myocytes mRNA level of prepro-atrial natriuretic peptide (pre-

pro-ANP), respectively (Fig. 6). The pretreatmentThe induction of prepro-ANP and -BNP mRNAs(24 h) with the farnesyltransferase inhibitor BMS-represent molecular events associated with cardiac191563 (25 l) inhibited norepinephrine- and en-hypertrophy. The treatment (24 h) of cardiac myo-

cytes with norepinephrine (1 l) or endothelin-1 dothelin-1-stimulated prepro-ANP mRNA ex-

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1135

Figure 5 The role of the extracellular signal-regulated kinase (ERK) in cardiac myocyte hypertrophy. (A) The treatmentof cardiac myocytes for 5 min with either norepinephrine (NE; 1 l) or endothelin-1 (ET-1; 10 n) increased ERKactivity, as assessed via the phosphorylation of the substrate myelin basic protein (MBP). (A,B) The pretreatment withthe farnesyltransferase inhibitor BMS-191563 (BMS; 25 l) completely inhibited the NE response, whereas a partialattenuation of ET-1-stimulated ERK activity was observed. (C) The pretreatment with the PD098059, a pharmacologicalagent which inhibits the upstream activator of ERK, the mitogen-activated protein kinase kinase MEK, did not attenuateeither NE- or ET-1-stimulated 3H-leucine uptake in cardiac myocytes. (∗) denotes P<0.01 v NE or ET-1.

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A. Calderone et al.1136

Figure 6 The regulation of prepro-atrial natriuretic peptide (prepro-ANP) and -brain natriuretic peptide (prepro-BNP)mRNA expression by the farnesyltransferase inhibitor BMS-191563 and the calmodulin inhibitor W7 in cardiacmyocytes. (A) A 24 h treatment with either norepinephrine (NE; 1 l) or endothelin-1 (ET-1; 10 n) increased thesteady-state mRNA levels of both prepro-ANP, and prepro-BNP. (A,B) The pretreatment with the farnesyltransferaseinhibitor BMS-191563 (BMS; 25 l) decreased NE- and ET-1-mediated expression of the mRNA transcripts. In parallel,the pretreatment with the calmodulin inhibitor W7 (5 l) attenuated NE-mediated expression of prepro-ANP andprepro-BNP mRNA transcripts. The steady-state mRNA levels of prepro-ANP and prepro-BNP were normalized toGAPDH mRNA, (∗) denotes P<0.05 v ET-1, and (∗∗) denotes P<0.01 v NE.

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1137

pression by 94±3% (n=4; P<0.001) and 65±11% well characterized that the prenylation of the cyst-eine residue of the CAAX motif located on the C-(n=4; P<0.05), respectively (Fig. 6). Moreover,

analogous to BMS-191563, the treatment of cardiac terminus of ras by the enzyme farnesyltransferaserepresents an essential postranslational modi-myocytes with the calmodulin inhibitor W7 (5 l)

attenuated norepinephrine-stimulated prepro-ANP fication required for the translocation of ras tothe plasma membrane, and subsequent biologicalmRNA expression by 66±23% (n=4; P<0.05)

(Fig. 6). In parallel experiments, norepinephrine and activity.32 This essential post-translational modi-fication has been exploited via the synthesis ofendothelin-1 stimulation (24 h) of cardiac myocytes

caused a 4.7±1.5-fold and 2.6±0.5-fold increase farnesyltransferase inhibitors which have provento be successful therapeutic agents to combat thein the steady-state mRNA level of prepro-brain

natriuretic peptide (prepro-BNP), respectively (Fig. malignant growth of ras-dependent tumor cells.33,34

In the present study, the treatment of neonatal6). The pretreatment (24 h) with BMS-191563(25 l) inhibited norepinephrine- and endothelin- rat cardiac myocytes with 25 l BMS-191563

caused a 42% decrease in the level of farnesylated1-stimulated expression of prepro-BNP mRNA by81±11% (n=3; P<0.005) and 61±20% (n=2), ras in the particulate fraction, and a concomitant

increase in unfarnesylated ras in the cytosolic frac-respectively (Fig. 6). Analogous to BMS-191563,the treatment of cardiac myocytes with W7 (5 l) tion. The reduction of particulate farnesylated ras

by BMS-191563 was associated with the at-attenuated norepinephrine-mediated expression ofprepro-BNP mRNA by 62±12% (n=3; P<0.01) tenuation of norepinephrine-mediated cardiac myo-

cyte hypertrophy and the expression of prepro-ANP(Fig. 6).and -BNP mRNAs. Likewise, endothelin-1 mediated3H-leucine uptake, and gene expression were alsosignificantly reduced by BMS-191563, albeit to alesser extent, as compared to norepinephrine. TheseDiscussiondata suggest ras-dependent signaling pathwayscontribute at least in part to norepinephrine- andThe increase in cardiac myocyte cell size, re-

organization of contracile proteins into sarcomeric endothelin-1-stimulated cardiac myocyte hyper-trophy and natriuretic peptide mRNA expression.structures, and the concomitant induction of vent-

ricular atrial natriuretic peptide (ANP) and brain The serine/threonine extracellular signal-reg-ulated kinase (ERK) is a putative downstream targetnatriuretic peptide (BNP) mRNAs represent con-

served features of the hypertrophic response to a1- of ras, and both endothelin-1 and norepinephrinetreatment of neonatal rat cardiac myocytes haveadrenergic agonists.6,10,21,27 Both in vivo and in vitro

studies have implicated ras as an integral trans- been shown to increase ras-GTP loading, and stimu-late ERK activity.10,28,35 Interestingly, despite aducer involved in the expression of these phenotypic

events.The direct injection of oncogenic Hras in- quantitatively similar magnitude of ras-GTP load-ing, the capacity to recruit the ERK pathway wascreased myocyte cell size, and the transcriptional

expression of both ANP- and BNP-promoter con- greater for endothelin-1 v a1-adrenergic agonists.28

Consistent with this observation, the present studystructs, albeit the reorganization of contractile pro-teins into sarcomeric structures, a hallmark feature has demonstrated endothelin-1 stimulated ERK ac-

tivity was >4-fold greater than norepinephrine.of cardiac myocyte hypertrophy, was not ubi-quitously observed.11–13,29 Evidence to support ras BMS-191563 treatment of cardiac myocytes in-

hibited norepinephrine- and endothelin-1 mediatedin the hypertrophic response of the a1-adrenergicreceptor includes an increase in ras-GTP loading, ERK activation, thereby confirming ras as an up-

stream activator of ERK, and the specificity of actionand the partial transcriptional inhibition of ANPfollowing the introduction of a dominant negative of the farnesyltransferase inhibitor. Moreover, ras-

dependent ERK activation may represent a hy-ras mutant.10,12,28 Recently, interleukin-1b-me-diated expression of a BNP-promoter construct was pertrophic signaling event. However, the treatment

of cardiac myocytes with PD098059,26 a phar-also shown to be ras-dependent.30 By contrast,despite the induction of a qualitatively similar hy- macological agent which inhibits the upstream ac-

tivator of ERK, the mitogen-activated protein kinasepertrophic phenotype, relatively little is known ofthe functional role of ras following the exposure of kinase (MEK), did not attenuate either no-

repinephrine or endothelin-1 stimulated 3H-leucinecardiac myocytes to the peptide endothelin-1.31

To assess the role of ras, a pharmacological ap- uptake. These findings are consistent with severalprevious studies demonstrating the lack of in-proach was employed utilizing the farne-

syltransferase inhibitor BMS-191563. It has been volvement of ERK in cardiac hypertrophy in re-

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A. Calderone et al.1138

sponse to a1-adrenergic agonists and endothelin- tenuated norepinephrine-stimulated 3H-leuine up-take, and caused a concomitant decrease in the1.36–38

The present study has demonstrated BMS- steady-state mRNA levels of prepro-ANP, and pre-pro-BNP. Taken together, these data underscore a191563 treatment of neonatal rat cardiac myocytes

inhibited ras farnesylation, which coincided with role of calmodulin in the hypertrophic response ofnorepinephrine. By contrast, W7 treatment did notthe subsequent attenuation of the norepinephrine,

and to a lesser extent, endothelin-1 mediated hy- alter the hypertrophic response of endothelin-1,despite the recruitment of the calcium mobilizingpertrophic response. However, based on the ob-

servation that ras-GTP loading was similar between second messenger inositol 1,4,5-trisphosphate. Thelack of involvement of the Ca2+/calmodulin path-endothelin-1 and a1-adrenergic agonists in neonatal

rat cardiac myocytes,28 we had anticipated a similar way in the endothelin-1 hypertrophic response isnot totally unexpected. Numerous G-proteinlevel of inhibition by BMS-191563. However, the

level of ras-GTP loading does not necessarily reflect coupled receptors have been shown to mobilizecalcium, albeit the recruitment of ubiquitous down-the magnitude of activation of putative downstream

targets. As previously documented,28,39 and ob- stream effector molecules are not necessarily cal-cium-dependent. The exposure of neonatal ratserved in the present study, ERK activation was

significantly greater in response to endothelin-1, as cardiac myocytes to either norepinephrine, iso-proterenol, angiotensin II, or endothelin-1 in-compared to norepinephrine. Moreover, endothelin-

1 stimulated inositol 1,4,5-trisphosphate pro- creased intracellular calcium, albeit onlyangiotensin II- and isoproterenol-stimulated ERKduction was found to be>3-fold more potent than

norepinephrine. Taken together, these data suggest activity were calcium-dependent.31,35,40–43 Con-sequently, these data demonstrate an additionaldespite the recruitment of common signaling path-

ways, important quantitative differences in the complexity in the signaling pathways coupled tothe recruitment of various effector molecules, de-magnitude of activation are evident between no-

repinephrine and endothelin-1. This difference may spite the activation of common second messengerpathways.be explained in part via a stimulus-specific re-

cruitment of distinct signaling events. Indeed, a The present study has demonstrated both ras-and calmodulin-dependent pathways contribute todisparate pattern of protein kinase C isoform ac-

tivation has been observed in neonatal rat cardiac the hypertrophic response of norepinephrine. In-terestingly, several reports support an interactionmyocytes following norepinephrine and endothelin-

1 treatment.39 Collectively, these observations sug- between these two pathways. In rat vascularsmooth muscle cells, angiotensin II mediated re-gest the partial attenuation of endothelin-1 stim-

ulated cardiac myocyte hypertrophy and natriuretic cruitment of the small GTP-binding protein ras andsubsequent activation of ERK in occurred via apeptide mRNA expression by BMS-191563 may

reflect in part the recruitment of an alternative ras- Ca2+/calmodulin dependent pathway.44 Moreover,in neonatal rat cardiac myocytes, the treatment ofindependent signaling pathway(s).

Several studies have demonstrated elevated in- cardiac myocytes with the Na+-K+ATPase inhibitoruoabain was shown to increase ERK activity andtracellular calcium promotes myocyte growth and

gene expression in part via the recruitment of the induce ANP expression in part via Ca2+/cal-modulin-dependent activation of ras.17 Collectively,calcium binding protein calmodulin. Specifically,

transgenic mice overexpressing calmodulin de- these observations suggest the Ca2+/calmodulinand ras-dependent pathways may act in a co-veloped cardiac hypertrophy, and the over-

expression of the calmodulin-dependent effector ordinated fashion to facilitate the hypertrophic re-sponse of a1-adrenergic receptors.molecules, calmodulin kinase or calcinuerin-de-

pendent transacting factor NF-AT3 (nuclear factors Farnesyltransferase inhibitors have been suc-cessfully employed to combat ras-dependent growthof activated T cells) have been linked to ANP and

BNP expression.17–20 Lastly, a pharmacological of numerous cell types, and in the present study,the treatment of neonatal rat cardiac myocytesstudy by Sei and colleagues demonstrated the cal-

modulin antagonist W7 suppressed ANP protein with BMS-191563 attenuated norepinephrine-, andto a lesser extent, endothelin-1-stimulated proteinexpression following phenylephrine treatment of

neonatal rat cardiac myocytes.15 However, the func- synthesis and natriuretic peptide mRNA expression.Collectively, these data underscore a role of thetional role of calmodulin in the hypertrophic growth

response per se, and its recruitment by physiological small GTP-binding protein ras in the hypertrophicresponse of cardiac myocytes to at least two G-relevant hypertrophic stimuli remains undefined.

In the present study, the treatment with W7 at- protein coupled receptors. Likewise, the calmodulin

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Farnesyltransferase Inhibitor Attenuates Myocyte Hypertrophy 1139

9. P TG, S MD. Growth factors, proto-antagonist W7 attenuated norepinephrine-stim-oncogenes, and plasticity of the cardiac phenotype.ulated cardiac myocyte hypertrophy, whereas en-Annu Rev Physiol 1991; 53: 179–200.

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cardiac muscle cell hypertrophy by inhibiting ac-endothelin-1 stimulated cardiac myocyte hyper-tivation of the Ras-MAP kinase signaling pathway.trophy, highlights the involvement of alternativeBiochem Biophys Res Commun 1994; 202: 1586–ras-independent hypertrophic signaling events, and1591.

their recruitment appears to be stimulus-specific. 11. H WA, T A. Ras and Rho are requiredLastly, an intrinsic role of ras has been identified for Gaq-induced hypertrophic gene expression in

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12. T A, T J, C S-Y, P S, S-latter observation and the findings of the present HE, F JR, C KR. Hras-dependent

study, the use of farnesyltransferase inhibitors alone pathways can activate morphological and geneticor in conjunction with other drugs may represent markers of cardiac muscle cell hypertrophy. J Biol

Chem 1993; 268: 2244–2249.a potentially important therapeutic approach in13. F SJ, G-B J, S PH. Oncogenictreating various cardiovascular disease states.

src, raf, and ras stimulate a hypertrophic pattern ofgene expression and increase cell size in neonatalrat ventricular myocytes. J Biol Chem 1998; 273:18146–18152.Acknowledgments

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