Age-related differences in the expression of proto-oncogene and contractile protein genes in response to pressure overload in the rat myocardium.

Age-related differences in the expression of proto-oncogene and contractile protein genes in response to pressure overload in the rat myocardium.

T Takahashi, … , W Grossman, S Izumo

J Clin Invest. 1992;89(3):939-946. https://doi.org/10.1172/JCI115675.

Cardiac adaptation to hemodynamic stress involves both quantitative (hypertrophy) and qualitative (pattern of gene expression) changes. Our previous studies have shown that advancing age in the rat is associated with diminished capacity to develop left ventricular hypertrophy in response to either ascending aortic constriction (AoC). In this study, we examined whether the expression of protooncogenes and contractile protein genes in response to AoC differs between adult (9-mo-old) and old (18-mo-old) rats. RNA was isolated from the left ventricles of AoC animals of both age groups subjected to a similar hemodynamic stress. Immediately after AoC, the levels of the ventricular expression of c-fos and c- jun protooncogenes were markedly lower in the old rats than in the adult animals. 5 d after the operation, the ratio of beta- to alpha-myosin heavy chain mRNAs increased significantly after AoC in both age groups. In contrast, AoC was associated with a marked reduction in the levels of mRNAs encoding sarcoplasmic reticulum Ca(2+)-ATPase (by 69%) and cardiac calsequestrin (by 49%) in the old rats but not in the adults. The mRNAs encoding atrial natriuretic factor and skeletal alpha-actin increased in response to AoC only in the adult rats. There were no significant differences in

expression of the cardiac alpha-actin mRNA among the experimental groups. These data suggest that (a) the expression of protooncogenes in response to […]

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Age-related Differences in the Expression of Proto-oncogene and Contractile Protein Genes in Response to Pressure Overload in the Rat Myocardium

ToshiyukiTakahashi,** HeribertSchunkert,t Shogen Isoyama,tJeanne Y.

Wei,:t

Bernardo

Nadal-Ginard,11

William

Grossman,*

andSeigoIzumo**

*MolecularMedicine UnitandDepartment

of

Medicine

(kCardiovascular

^and

OGerontology Divisions),

^BethIsrael

Hospital,

I"Department of Cardiology, Children'sHospital, andDepartments ofMedicine andPediatrics, Harvard Medical School, Boston, Massachusetts 02215

Abstract Introduction

Cardiacadaptationtohemodynamicstressinvolves bothquan- titative

(hypertrophy)

andqualitative (pattern

of

gene expres- sion) changes. Ourprevious studies have shown that

advancing

agein theratis associated with diminishedcapacitytodevelop left ventricular hypertrophy in response to either ascending aortic constriction (AoC). In this study,weexamined whether theexpression ofprotooncogenesandcontractile

protein

genes in response to AoCdiffers between adult (9-mo-old)and old (18-mo-old)rats. RNA wasisolated from the left ventricles of AoC animalsof bothage groupssubjectedto asimilarhemody- namicstress.Immediately after AoC,the levelsoftheventricu- larexpressionof

c-fos

andc-junprotooncogeneswere markedly lower inthe old rats than inthe adultanimals. 5 dafter the

operation,

the ratio of,- to

a-myosin heavy

chain mRNAs increased

significantly

after AoC in bothage groups. Incon- trast,AoCwasassociated with^amarkedreduction in the levels of mRNAs

encoding sarcoplasmic

reticulum

Ca2"-ATPase (by

69%) and cardiac calsequestrin

(by 49%)

in the oldratsbutnot intheadults. The mRNAs

encoding

atrial natriuretic factor and skeletala-actin increasedin responsetoAoC

only

in the adult rats.Therewere no

significant

differencesin

expression

of the cardiac a-actin mRNAamongthe

experimental

groups.These datasuggestthat

(a)

the

expression

ofprotooncogenes inre- sponse to acute pressureoverload is

significantly

reduced inthe agedrats and

(b)

thepattern of

expression

of the contractile proteingeneinresponse toAoC in the oldrats

differs qualita- tively

aswellas

quantitatively

fromthatinyounger animals.

These age-related differences may play a role in the higher frequency of heart failure in the aged during hemodynamic stress.

(J. Clin.

Invest. 1992.

89:939-946.) Key

words:actin . myosin heavy chain*calsequestrin*

Ca2+-ATPase

^-atrial natri- uretic factor

Partof this work was presented at the 62nd Annual Scientific Sessions of the American Heart Association on 15 November 1989, in New Orleans, LA. Portions of this work have been published in abstract form (1989. Circulation. 80 (Suppl.II):II-457).

Address correspondence to S. Izumo, M.D., Molecular Medicine Unit,Beth IsraelHospital,330Brookline Avenue, Boston, MA 02215.

Receivedfor publication22February 1991 and in

revisedform

28 October 1991.

Thephenomenon

of

senescenceis of fundamental

importance for

the function of cardiac muscle

because,

likeneurons, car-

diac

myocytes are

terminally differentiated

cells and their life spanisas

long

asthat of the whole

organism.

Since adultcar-

diac

cellsareunableto

divide,

the

ability

of the heartto

adapt

toincreased

hemodynamic load, either

from

hypertension,

val- vular heart

disease,

or loss

of myocardium

dueto ischemic heartdisease, is critically dependentonits

capacity

todevelop hypertrophy(1-3). When

functional

demands exceed

adaptive

limits, heart

failure

ensues(3, 4).

It

is

well knownthat, when

subjected

to

similar hemody- namic

stress,old

patients

develop heart failuremore

frequently

than

their

youngercohorts

(5, 6).

This

higher

incidence ofheart

failure

in old

patients might

be dueto a

diminished capacity for cardiac hypertrophy

inresponse toa

given

stress. It hasbeen shown that theextent

of cardiac hypertrophy

induced

by

exer- cise is

substantially

less in thesenescentthan in youngadult

animals (7).

Our

previous studies

have

confirmed

that theca-

pacity for left ventricular (LV)' hypertrophy

in response to pressure or volume overloaddiminishes with

advancing

age

(8, 9).

Cardiac adaptations

to

hemodynamic

stress also

involve

alteredpattern

of

gene

expression. Changes

in

expression of

^a variety

ofcardiac

genesinresponse tooverload have beensum-

marized

astheacute

induction of

protooncogenes,

followed

by the

reinduction

of fetal

isoforms ofcontractile proteins (10-12)

such as

0-myosin ^heavy

^chain

^{(MHC) (13,}

14) and skeletal a-actin (10, 15).In

addition,

pressureoverload has been shown tomarkedly

increase ventricular

mRNAlevels

of atrial natri-

uretic

factor (ANF) (10, 16,

17) andto

significantly

decrease themRNAlevels

of sarcoplasmic

reticulum

(SR) Ca2+-ATPase (18-20).

Thus,

hemodynamic

stresschanges the levels

of

these mRNA

species similar

tothose observedduring the fetalstage (10,

11,

18).

Normal myocardial aging also involvesboth quantitative and

qualitative

alterations (21, 22). Morphologically, intersti-

tial fibrosis

andmyocytehypertrophy havebeen observedin the

left ventricles of

normalaged rats(22, 23). Physiological studies of^LVpapillarymusclepreparationshave revealed that theduration oftension and the timeto half-relaxationwere prolonged inaged rats comparedwithin their younger cohort (21, 24).Theprolongation ofmyocardial contraction has been 1.Abbreviationsused in this paper: AoC, ascending aortic constriction;

ANF, atrial natriuretic factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MHC, myosin heavy chain; SR, sarcoplasmic retic- ulum.

J.Clin. Invest.

© The AmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/92/03/0939/08 $2.00 Volume 89, March 1992, 939-946

attributedto adecline of Ca2+-activated myosin-ATPase activ- ity,secondarytoadecrease inthepercentageofa-MHCorV1 isozyme (25, 26). The abnormal myocardial relaxation has beenthoughttoreflect age-related decreases in Ca2+-uptakeby theSR (27, 28).

In the present study, we asked the following questions.

First,canthe agingprocessaffectchanges in cardiac gene ex- pression inresponse toaortic constriction (AoC)? Second, do qualitative changes in thepatternofgeneexpressionoccurin response to an ascending AoC in the old animals, in spite ofthe absence of quantitative hypertrophicresponses(8)? Thesetwo questionsmaybeimportant because theymaybe related both tofundamental mechanisms concerning the effects of agingon regulation ofgeneexpression andtoclinical problemsconcern- ing the age-related differences in the frequency of heart failure.

To answer these questions,weexamined expression of genes encodingtwo protooncogenes(c-fos and

c-jun),

two SRpro- teins (SR Ca2+-ATPase and cardiac calsequestrin), two iso- formsof sarcomeric proteins (skeletal and cardiac a-actins and a-and 13-MHC) and a secreted protein (ANF) in the LV myo- cardiumof the adult(9-mo-old) and old (18-mo-old)ratswith orwithout pressure overload.

Methods Materials

Twoage groups(9and 18 moold) ofmaleFisher344 rats(Harlan Sprague Dawley, Inc.,Indianapolis,IN)wereobtainedunder contract with the NationalInstitute of Aging.Averagelifespan of the malesof thiscolony is ^- 24 mo. The ratsofthisspeciesdo notgainbodyweight between9and 18 moofage,and their bloodpressure does notchange during this time period (8).

Operative procedures

Operative procedures forproducinganascendingAoCweredescribed indetail previously (8). Briefly, eachrat wasanesthetized either with brevital(50 mg/kgi.p.)orwithmethohexital sodium(60 mg/kg i.p.).

The rats wereintubatedand respirationwas controlledbyarodent ventilator (model683;HarvardApparatus,SouthNatick, MA) with roomair.Theleftthorax wasopenedatthethird intercostalspaceto expose theascendingaorta. Theascendingaortawascarefully dissected free from the pulmonaryarteryandsurroundingtissues,and asurgical thread(1-0 silk)wasdrawnundertheascending^aorta. A 16-gauge needle(1.6mmo.d.)wasplaced alongsidetheascendingaorta, and the aorta and needle weretiedtightly togetherwith the thread. Theneedle wasthenremoved, leaving the ascendingaortaconstrictedto adiame- terof 1.6mm.The thoraxwasclosed withasilksuture, whereasthe lungswereinflated withpositive end-expiratorypressure. Forsham- operatedgroups, theidenticalprocedurewasperformed,except that induction ofAoC was notdone.Asreported previously (8), operative mortalities of9- and 18-mo-oldratswith AoC^were15and23%,respec- tively.

RNA measurement and analyses

RNAanalyseswereperformedin groupsof56 animals(n⁼28 each for 9- and 18-mo-oldrats)that hadundergonetheoperations^described above, creatingAoC(n⁼34)orperforming^ashamoperation (n⁼22).

Rats were killed at30,45,60, 90,and 180 minafterAoCforthestudy ofprotooncogenes^andat5d for thestudyof contractileproteingene expression.Theleft ventriclewascarefullydissected from the atria and right ventricleandstored inliquid nitrogenuntiluse. Total cellular RNA was extractedby the guanidinium-hot phenol method. The RNAconcentration in the LVmyocardiumwasdetermined byspectro- photometry, with the absorbance at260 nm.Theratio of^theabsor- banceat280nmwas >1.9 in all thesamples.

RNA blot analysis. 20-jgsamples of total cellular RNA from each animal were sizefractionatedbyelectrophoresis^{on a 1.0%}agarose-for- maldehydegel andtransferredto nylon ornitrocellulose filters. The filters were prehybridized in the solution mix (containing 50% form- amide, 5Xstandard saline citrate [SSC; lX ⁼0.15 M NaCl, 0.015 M sodium citrate], 5X Denhardt'ssolution, 0.2% SDS, 0.025 M sodium phosphate, and 250,ug/ml^calfthymus DNA) at420C for 2 2 h and hybridized with specific DNA probes in the same solution mix plus 10%dextran sulfate, at420C,for 16 h. For the 18-S rRNA oligonucleo- tide probe,double-distilledwater was substituted forformamide. At the endofhybridization,the filters were washed serially, with the final wash in

0.lX

SSC-0.2% SDS at650C^{for 15-90} min,except for the skeletal a-actin probe (in 0.5X SSC-0.1%SDS at550Cfor 15min)and the 18-S rRNA probe(in 3XSSC at roomtemperaturefor 30min).

DNA probes used in this study were as follows: (a) c-fos: a 2.1-kbEcoRI fragmentofthe mousec-foscDNA clone pGEM-fos (a generous gift fromDr. M. E.Greenberg);(b) c-jun: a 2.1-kb

fragment

of the mouse c-jun cDNA (29); (c) SRCa2+-ATPase:a0.7-kbPstI fiagmentgener- ated from the carboxy terminal and 3' untranslated region of the cDNA clone pCA, specific for the rabbit cardiac-slow twitch skeletal muscle SRCa2+-ATPase(30);(d)cardiac calsequestrin: a 1.9-kb EcoRI frag- mentfrom a cDNA clone IC3A, containing the entire coding region and 3' untranslated region of a canine cardiaccalsequestrin (31); (e) ANF: a synthetic 84-nucleotide-longoligonucleotide complementary to the entire codingsequenceofthe ratANF(32); (f) skeletal a-actin: a synthetic oligonucleotide complementarytothe first 57 nucleotides of the 3'untranslated region ofthe mouse skeletal a-actin cDNA (33); (g) cardiac a-actin:asynthetic oligonucleotidecomplementary to the first 55 nucleotides of the 5'untranslatedregion of the mouse actin cDNA (33);(h) glyceraldehyde-3-phosphate dehydrogenase(GAPDH): a 1.3- kbPstIfragment from cDNApUC-GAPDH13,containing the entire codingregion and a partof3'untranslated region of the rat GAPDH (34); and(i) ^18-SrRNA: a syntheticoligonucleotide (5'-ACGGTAT- CTGATCGTCTTCGA-3') complementaryto the rat 18-S rRNA (35).

The cDNA probes were labeled by random priming method with [32P]dCTP(3,000Ci/mmol,New England Nuclear, Boston, MA) and thesynthetic oligonucleotide^probeswithT4polynucleotidekinase and

[32P]yATP

(3,000 Ci/mmol, New England Nuclear) (36).

The blots were exposed on x-ray films with intensifying screens for 1-7 d at -80'C. Relativeamounts ofeach mRNA weredeterminedby laser densitometry in thelinearresponse range of the x-ray films. The densitometricscores ofspecificmRNAs were "normalized" by that of GAPDH mRNA,which encodesaconstitutively expressed glycolytic enzyme,as an internal control.Normalizationby18-S ribosomal RNA signalsor by thecardiac a-actinsignals produced similar results. The mean value of the normalized scores of mRNA from sham-operated, adult(9-mo-old) animals wasarbitrarily determinedas 1.0 for each mRNA species.

SInuclease mapping.S1mapping analysis was performed as previ- ouslydescribed(14).Briefly,20

jig

oftotal RNA was hybridized in 80%

formamide at

420C

for 16 h with a 3' end-labeled, single-stranded probe, generated from the 3' endPstIfragment of NB3, a cDNA spe- cific for therat

13-MHC

(14). This347-nucleotide-longprobecontains 180nucleotidesofcommon coding sequence at the carboxy terminal of the a- and

fl-MHC,

ⁱⁿaddition to the entire 3' untranslated sequence of the

fl-MHC

gene, whichdivergescompletely from the a-MHC gene.

Thisprobe alsocontains43nucleotides of oligo (dT) tails.S1 nuclease digestionwasdone with 150 U of enzyme (New England Nuclear) and thedigestionproducts were size-separated on 7%polyacrylamide-8.3 Murea-sequencinggel. The gel was dried and exposed on an x-ray film at-80°C as mentioned above. Therelativeamountsofa-and,B-MHC mRNAs were quantitated by laser densitometry in the linear rangeof the x-ray film.

Statistical analyses

All data are expressed as means±SEM. The statistical significance of differences in meanvalues between two groups^wasassessedby ^the

unpairedStudent's t-test withBonferroni's correctionorFisher'sexact testifoverall significancewasdemonstratedby analysis of variance (37).Significancewasacceptedat P<0.05 level.

Results

The present study is a

direct

extension of our previous study concerning the

hemodynamic

andmorphological changes in response to AoC in the rat LV myocardium (8). Table I summa- rizes the resultsofthe hemodynamic and

morphological

param- eters in this model. The degrees ofthe severity of pressure over- load

imposed

on the

left

ventricles were

similar

in both age groups (LV peak systolic pressure in mmHg: sham 91±3 and AoC 127±13

for

theadult; sham 95±9 and AoC 124±14 for theold).

This

pressure overloadcausedamoderatebut signifi- cantLV

hypertrophic

response

(23%

increase in LV dry weight in 4 wk) in the adult rats but no responses in the old rats. At histological level, the mean value of myocyte

width

in the sham-operated old

animals

wasmuchlarger than that in the

sham-operated

adults

(29.2±1.4

vs. 20.4±0.8 u). AoC in- creasedthe mean value of myocyte

width significantly (from

20.4±0.8 to 25.9±0.9

Mm)

in the adult rats but

did

not cause a further increase in this index in the old. These age-related dif-

ferences

in LV

hypertrophic

responsestoAoC were also evi- dent in the LV

myocardial

RNA

concentration, which

also

increased significantly (21%

over

sham)

in the

adult,

but

by

only 4%in the old.

Thus,

atthe moderate pressure overload used inthis model,LVhypertrophy occurred in the adult but not in the old rats.

In

this

study, we asked whether the

aging

process

affects changes

incardiac gene

expression

in responsetoAoC, in

spite

of the absence of

quantitative hypertrophic

response in the old

animals. First,

we examined

expression of

two

"immediate early"

genes,

c-fos

and c-jun protooncogenes, in response to acute pressure overload. As shown in

Fig.

1,

c-fos,

mRNA was markedly

induced

at90 min

after

AoC in9-mo-old rats, but

its expression

was

significantly

reduced in 18-mo-old

animals.

The

induction

of

c-fos

was

transient, inasmuch

as

its

level

of expression

returned tonearthe

baseline

by 180 min after AoC.

This

age-related

difference

in

c-fos expression

was also

ob-

served when the

animals

were

killed

at

earlier time points

(30, 45, and 60 min) after AoC (data not shown).

Expression of

c-jun in response to AoC was also reduced in the old rats com- pared with the adults at 90 min

after

AoC. Thus, the old hearts

appearto haveanattenuated nuclear responseto acutepres- sure

overload,

asdetermined

by

the two-protooncogene induc- tion.

Next,

weexamined

expression

of various contractile pro- tein genes in responsetoAoC.One ofthe well-studied biochem- ical markers of

hypertrophy

in theratisincreased

fl-MHC/a-

MHC ratio. To determine the levels ofa-and

f3-MHC ^mRNAs,

we

performed

S l nuclease mapping

analysis (Fig.

2). SI

diges-

tion

produced

twoprotected bands of 180 and 304

nucleotides,

correspondingto a-and

fl-MHC

mRNAs, respectively. In 9- mo-old animals, the level of,B-MHC mRNA was

significantly

higher than in young (2-3-mo-old) animals (14). The ratio of (3/a MHC mRNAswassimilar between the 9- and 18-mo-old sham-operatedrats.In responsetoAoC, the (3/a ratio increased to asimilar

degree

inboth age groups. In the adult rats, this increase in the (3/a ratio seemedtobe achieved primarily byan increase in the (3-MHC mRNAs, whereas in the old rats the same increase appearedtobe achieved

primarily

byadecrease in the a-MHC mRNA. Thus, in response to pressure overload, the (3/a ratio increased in both age groups, but possibly

by

different mechanisms.

Wealso examined changes in the level ofthe SR Ca2+-ATP- ase mRNA in response to AoC. Northern blot analysis has detectedan mRNA

species

of 4kb in the rat LV myocar- dium (Fig. 3, top row). Densitometric analysis of the hybridiz- ing signal from each animal revealed that the level of the SR Ca2+-ATPase mRNA, corrected by GAPDH signals, remained unchanged after AoC in the adult rats but decreased markedly (by 69%, P < 0.01) in the old rats (Fig. 4, left). Slot blot analysis of RNA yielded a very similar result (data not shown). Similar results were also obtained when the SR

Ca2+-ATPase

signals were corrected

either

by the

a-cardiac actin signals

orby the

18-Sribosomal signals (data not shown).

The aging process alone, at least

until

age 18 mo,

did

not alter expression ofthe SR Ca2+-ATPase gene, because therewas nosignificant difference in the level of this mRNA

species

be- tweenthe

sham-operated

adult and oldrats.

However,

atvery advanced age (30 mo), the SR Ca2+-ATPase mRNAwas re- ported to be decreased by 60% compared with young (4-mo- old) animals (38).

We then analyzed

expression

of

cardiac

calsequestrin, a highcapacity and medium affinity

Ca2`-binding

protein local- izedatthe

terminal cisternae portion of cardiac

SR

(31). Little

is known concerning regulation of the cardiac calsequestrin TableL Changes in Hemodynamic and Morphological Parameters and RNA Concentration in the LVMyocardium

in Response to AoC intheAdult (9-mo-old) and old (18-mo-old)Rats

Adult Old

Sham AoC Sham AoC

LVPSP (mmHg) 91±3 127±13* 95±9 124±14t

LVEDP (mmHg) 4.6±0.8 5.6±0.9 5.11±.0 6.0±0.6

LVdryweight/bodyweight (mg/g) 0.47±0.01

0.58±0.03*

0.49±0.01 0.49±0.01

LVdryweight/tibiallength (mg/cm) 39±1

44±2*

39±1 39±2

Myocyte width

(,Mm)

20.4±0.8

25.9±0.9*

29.2±0.6 29.2±1.4

RNAConcentration(gg/g^{wetwt) 963±22 1,168±31t} 980±10 1,021±16

Dataaremeans±SE.^*P <0.05,* P <0.01 comparedwith shamof the same age group. See reference 8 for more detailed hemodynamic and morphologicaldata. LV,left ventricular; PSP and EDP, peak systolic and end-diastolic pressures, respectively. Adults were 9 mo and old rats were18 mo old.

A ADULT OLD

(9M) (isM)

I II I

c C .

Eg E

E~^iE

E~

c0O0

⁰⁰⁰

co 0 ⁰ .c ^{0 0}

c ^o o ) 0 (

en < < en < <

c-fos -Mm_ t

c-jun

is ^1'

^_

GAPDH

4040a_ ⁴

^_

c-fos C c-jun

U 0

E

0

00(^A.0

it) 0 N aE

z0

Adult Old

(9month) (18month)

Adult Old

(9month) (18month)

Figure1.Expression ofc-fosand c-jun mRNA inresponseto acutepressureoverload. (A) RNA blothybridization.Thesameblotwashybridized withc-fos, c-jun, andGAPDH cDNA probes.(B and^C)Normalized densitometricscores.Thesignals ofc-fosandc-junwerecorrected by those of GAPDH.The numbers of samplesare n⁼ 3for sham in eachgroup; n⁼6for sham in eachgroup; n⁼6for AoC in eachgroup. .,

Sham; n,AoC90min;m,AoC 180min.

gene, particularly in pathologicstates suchas cardiac hyper- trophy and failure. Northern blot analysis (Fig. 3, 2nd row) indicated thatasingle cardiac calsequestrin mRNA species of 2.9kbwasexpressedintheratLVmyocardium. The level of cardiac calsequestrinmRNAexpression didnotchangeinre- sponsetoAoCinthe adultratsbut decreased significantly (by 49%, P< 0.05) inthe agedrats(Fig. 4, right). Therewas no

age-related differenceinexpressionof this SRproteingenein the sham-operated animals.

Fig. 5 shows the representative RNA blot analyses of mRNAs encoding ANF and skeletal and cardiac a-actinsinthe sham-operated and AoCrats of bothage groups, andFig. 6 summarizeschanges in the relative abundance of mRNAs by densitometric analysis. ByRNAblot analysis (Fig. 5,toprow), LV expression of ANF mRNA (- 1.0 kb)wasbarely detect- able. However, themean level ofANF mRNA increasedin

responsetoAoC in the adultrats.The level ofANF mRNAwas

already highinthesham-operatedold rats.AoC failedtocause

afurthersignificantincrease in the level of thismRNA inthe oldrats(Fig.6A).

Themeanlevel of skeletal a-actinmRNAincreased inre- sponsetoAoC in the adultrats(Fig. 5,2ndrow,andFig.6B).

In the old sham-operated rats, the level of skeletal a-actin mRNAwasnotdifferent from thatinthe adultsham-operated ratsand AoC didnotincrease expression of this mRNAspe-

cies. The level ofcardiac a-actin mRNA was not different amongtheexperimentalgroups.(Fig. 5,3rdrow,andFig.6C).

Discussion

This study demonstrated that thepattern ofexpressionofcar-

diacgene expressionin response to pressure overloaddiffers markedly between the adult and aged animals. Expressionof twoimmediateearlygenes,c-fosandc-jun,inresponsetoAoC is markedly attenuated in the oldrats.Incontrast,expression ofsomecardiacgenes(SRCa2+-ATPase,cardiaccalsequestrin, 942 Takahashi,Schunkert,Isoyama, Wei,Nadal-Ginard, Grossman,and Izumo

B

U

w

0

E0 Ca

e0S

so

a a

co

0N

aE

h.

z0

1.25

1.00 0.75 0.50 0.25

Adult Old (9 M) ⁽¹⁸M)

o

.0 0 1.2-

0 ¹

_t X @ ~~0 .8 ^;

a) 0.6,

01 0-4

_ _

0I ^{A c- -4} seen

0- Adult Old ^{a4HC- _}

(9 month) (18month)

Figure 2. Expression of the^a-and ,B-MHCmRNAsinthe LVmyo-

cardium.Slnuclease mapping analysiswasperformed witha3'end- labeled, single-strand probe,generatedfromthe3' end PstIfragment ofNB3, cDNA-specific for therat

fl-MHC

(3, 10). Each lanerepre-

sentsthepooled sample fromfiveanimals ofeach^group.Sl digestion createdtwoprotected bands of 180 and 304 nucleotides,correspond- ingtoa-and

fl-MHC

^mRNAs,respectively.Relativeamountsof the

a-and,B-MHC mRNAsweredeterminedby laserdensitometryand theratioof,3-MHCtoa-MHC(a/a)^wascalculated and shown inthe bargraph.The

#/a

ratioincreased similarly in both^{age groups}in

responsetoAoC.a,Sham; *, AoC.

0.2+.

E

0 z

SR Ca2+-ATPase

p<0..Di

_1.2

Calsequestrin

p< 0.05

j - _~06

[L~~~~tIi

⁰⁶⁴

0.2

Adult Old Adult Old

(9 month) (18 month) (9 month) (18 month) Figure4.Changesinthe levels ofmRNAsencoding theSR Ca2+-ATPase (left)andcardiaccalsequestrin (right)inresponseto AoC. The relative abundance of theSRCa2+-ATPasemRNA(nor- malized by the level of GAPDH mRNA) remained unchangedin

responsetoAoC in the adultrats,but decreased markedly (by 69%, P<0.01)intheoldrats.The level of cardiaccalsequestrinmRNA also decreasedsignificantly (by 49%,P^<0.05)afterAoC in the old ratsbutnotinthe adult.n= 5foreachgroup.IP^<0.01 adultvs.

oldanimals.a,Sham; *, AoC.

andMHCs) changedinresponsetoAoCintheoldrats, inspite of the absence of LVhypertrophic response inthisgroupof animals. On the other hand, the levels of mRNAs encoding ANFandskeletal a-actin didnotchangeafter AoC in the old animals. These datasuggestthatthere existsadissociation be-

Adult

Old

(9 M) (18 M)

En En

.M ⁰ r-0

W <~ Cflu(

tweenthequantitative (hypertrophy)andqualitative (patterns ofgeneexpression) responsesofthe LV myocardiumtothe hemodynamicstressandagingoncardiacgeneexpressionwere

complex and varied with individualgenesstudied,suggesting thatunderlying molecular mechanismsfor the alteredexpres-

sionof thesegenesmayalso becomplex.

Recent studieshaveshownthat senescent human cultured fibroblasts failedto proliferate in response to serum (39) or

platelet-derivedgrowth factor (40). Interestingly, expressionof

c-fos genefailedtoincreasebyserumstimulus in the senescent cells(39). These findingsarereminiscentofourresults because theoldmyocardium hadverydiminishedexpressionofc-fos

and c-jun and failed to develop hypertrophy in response to

Ca2+ -ATPase--- o I ⁱ _{Adult Old}

(9 M) (18 M) Calsequestrin-

EU

.m

⁰

= 0

en <:

GAPDH- 11

1 8S-*

Figure 3. Expression of mRNAsencodingtheSRCa2+-ATPaseand cardiaccalsequestrinin theratLVmyocardium.RNAblothybrid- izationwascarriedoutwith cDNA clonesspecificfor the rabbitcar- diac-slowtwitchskeletalSRCa2+-ATPase(27) andcaninecardiac calsequestrin(25).The SR

Ca2+-ATPase

cDNAprobe (pCA) hybrid- ized withanmRNAspecies of ^- 4kb. Thecardiac calsequestrin cDNAprobe(IC3A)producedasinglebandof ^- 2.9 kb. In contrast tothecanine heart(25),theadditionalbandof2.2kbwasnotde- tected with the ICA3 clone in theratLVmyocardium.The same blot washybridizedwith a cDNAforGAPDH (29) andasynthetic oligo- nucleotide for 18-S rRNA asinternalcontrols. Each lane represents thepooled sample from five animals ofeach group.Expression of the SRCa2+-ATPaseandcalsequestrinmRNAsdecreasedmarkedly in the old AoCanimalsbutremained unchangedin theadultanimals.

ANF

cm u on .40

urn

Skeletal- *

a-actin Cardiac- a-actin GAPDH-

Figure5.Representa- tive RNA blotanalysis of theexpressionof mRNAsencodingANF and skeletal and cardiac a-actins in therat LV myocardium. Each lane representsthepooled samplesoffiveanimals of eachgroup.Synthetic oligonucleotideswere

used forthehybridiza- tion. The^meanlevel of ANFmRNA(1.0kb in size)morethandoubled inresponsetoAoC in the adultrats. Incon- trast,ANFmRNAwas

alreadyhighⁱⁿthe LV myocardium fromthe oldsham-operated^rats and didnotincrease furtherafterAoC. Themeanlevel ofskeletal a-actinmRNA(1.8 kb) alsoelevatedby twofoldintheadult ratswithAoCbut showedno

changes in the old animals. The level of cardiac a-actinmRNA(1.8 kb)wasnotdifferentamongtheexperimentalgroups.

Age-related Differencesin CardiacGeneExpression 943 1.6

1.4 1.2 1

C 0.8

' 0.6 0.4 0.2

"HC-+

ANF

8

4)

E

0

U)

0.

00

0

-0N

E

0 z

7-

6

5-

4 3 2

0

Adult (9 month)

B

3.5

0

E

0

01

c00

0^- Us

o0

n

0

N

m E 0 z

3

2.5 I

2

1.5 |

Old

(18 month)

Skeletal a-Actin

IT

I3.. A.

0.5 1

0 -A-l

Adult Old

(9 month) ⁽¹⁸ month)

c Cardiac a-Actin

1.4

t _-

^1.2 t

4)

E

° 1.0

U)

CD)

ao 0.Bt

w 0.6

N

E 0.4j

To

z

0.2

0.0 L

T

Adult (9 month)

Old

(18 month)

Figure^6.Densitometricanalysis^ofthe levels ofmRNAsencoding

ANF(A)^andskeletal(B)^andcardiac a-actin(C)ⁱⁿthe LVmyocar- dium.Therelativeabundance of thespecific^mRNAspecies^{was nor-}

malizedby^{thelevel of}GAPDH mRNA in eachanimal.n⁼5 for eachgroup.^.,Sham;^.,AoC.

AoC. Suchsimilarity may imply that common mechanisms might underlie the agingprocessof the cellgrowthsystem in cardiocytes and other cell types. It should be noted,however, that at presentthere isnoformalproof that expressionofpro-

tooncogene iscausatively linkedtocardiac hypertrophy.Italso remains to bedeterminedwhether expression ofother immedi- ateearlygenesis alsoreduced inresponsetoAoC in the aged heart.

We usedFisher 344ratsbecause in this species bodyweight, heartweight, and bloodpressuredonotincrease from theadult stagetooldage.Incontrast,Sprague-Dawley andLong-Evans ratsdevelop age-related cardiac enlargement. Fisher 344 rats are also known to have significant age-related myocyte loss (22). Consistent with this finding, our histological study showed that themean value ofmyocytewidth inthe sham- operated old animalswassignificantly larger than that inthe adult sham-operated rats(29.2±0.6 vs. 20.4±0.8

tim).

^Thus,

advancingagealoneappearstocausehypertrophyatthecellu- lar,but not attheorgan,level to compensateformyocyteloss accompanyingtheagingprocess.

AoC increased themeanvalue ofmyocytewidth from 20to 26 ,uminthe adult ratsbutdidnotcause afurther increase in this indexin the old. On the otherhand, the biochemical pheno- type of the stressed myocardium of the old animals is very

similar tothat of theadvancedhypertrophy in theyoung(10- 20).Thisindicates that thesenescentmyocytesareabletosense

increased load andchangethe patternofgeneexpression. How- ever,theyareunable toincreasetheirsizefurther,atleastnotin

responsetothe moderatepressureoverload used in this study.

It ispossiblethatthesenescent myocytesmaybe approaching the limit in sizeduringthenormalagingprocess.Inthis regard, it is noteworthy that hypertrophied ventricles ofyoung rats (produced by AoC) had very diminished expression ofc-fos andc-juninresponseto acutepressureoverload in vitrocom-

paredwith those innonhypertrophied ventricles (41).

Analternativeinterpretationoftheage-related differences might be considered. It ispossible that proliferation of fibro- blastsistriggered morereadily by pressure overload inaged thaninadult rats.Dilution ofmyocyteRNAwithnonmyocyte RNA inthe total RNApreparation couldcreatethespurious appearance ofa decline in theabundance ofcertain mRNA species (Ca2+-ATPaseorcalsequestrin)and maskoverload-in- duced increasesinabundance ofotherspecies

(f3-MHC,

^skele-

talactin,c-fos, c-jun,orANF).This effectmaynotbedetected bythe GAPDH ^or 18-S RNAcontrol signals. However,itis highly unlikely that this is the sole cause of the age-related differences observed in cardiac geneexpression,because(a)the decrease in theCa2+-ATPaseandcalsequestrinmRNAs inthe aged rats with AoCwasevident evenwhen corrected bythe level of the cardiac actinmRNA,^amyocyte-specificgeneprod- uct; (b)theage-relateddifferencesinprotooncogene expression inresponsetoAoCwereobservedmuch earlier thanprolifera- tion of fibroblast could takeplace;and(c)thereisnoevidence for exuberantproliferationof fibroblasts sufficient todilutethe cardiac-specific mRNAsby ^- 50% in the old heart prepara- tions,since the total RNA content increasedby only^4%after AoCinthe oldrats.

Incontrasttoprevious studies(18-20), expression ofthe SRCa2+-ATPaseinourstudy^remainedunchangedin response toAoC in the adult rats. This is mostlikely^{due toa}moderate degree^ofpressure^overload(a^meanof33-mmHg^increasein

A

1

.~

T

LVsystolicpressure)used inourstudy.Ventriculardry

weight/

bodyweightratio increasedby^>40% in other studies( 18, 19), butby only 23% in ours. It has been demonstrated that the level of the SRCa2+-ATPase mRNA does not

change

in mild to

moderateLVhypertrophyin youngrats

(20). However,

more severepressure overloadwas notfeasible in thisstudy,because operativemortalitywasunacceptably high(> 50%)in the

aged

rats.

We examined expression of the cardiac genes

only

at mRNAlevel, and it is

possible

^thatsometranslationalorpost- translational controls maybe present in

regulation

^{of these} genes.However,

previous

studies have shown that the mRNA andprotein levels of the SR

Ca2+-ATPase (18, 20),

^ANF

(16),

and MHCs(14)

changed

ina

parallel

fashion in the LV myo- cardium subjected to pressure overload.

Although

yet^{to be} proven formally, it is likely that the

changes

in the mRNA levels in our

study

are

likely

^to be reflected to those of the

corresponding proteins.

At present, we do not know whether our

findings

^are

unique

to this

experimental

model or are more

general phe-

nomena.Ifwe canextrapolateour

findings

^tohuman

subjects,

the present

study

suggeststhe

possibility

that the diminished abilitytoinduceproto-oncogenes in responseto

hemodynamic

overload,aswellasthe

age-related

differences in cardiac gene

expression,

may

contribute

to the

higher frequency

of heart

failure

in old

patients

than inyoungcohorts

subjected

^tosimi- lar

hemodynamic

stress.

Although

an acute

obstruction

ofas-

cending

aorta is

extremely

rare in the clinical

setting,

other kinds ofacute

hemodynamic

overload, suchas acutemyocar-

dial infarction

and acute mitral

regurgitation,

arevery com- monin aged

patients.

Altered

expression

ofa

variety

of cardiac gene

expression might

^berelatedto

systolic

and diastolic

dys-

function, which is observed often in oldpatients.

Acknowledgments

We thank Dr.Yutaka Kagaya for his excellent surgical assistance, Dr.

R. D.Rosenberg for support, Dr.DavidH.MacLennanfor his kind gift of therabbit cardiac-slowtwitch skeletal SR

Ca2+-ATPase

cDNA, Dr.

M. E.Greenbergfor

c-fos

cDNA, Dr. M.Karinfor c-jun cDNA, and ElaineWuforhertechnicalassistance.

This work was supported in part by a grant from the American Federation forAging Research and Bayer Fund for Cardiovascular Re- searchtoS.Izumo.

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