• No results found

Reduced beta 1 receptor messenger RNA abundance in the failing human heart

N/A
N/A
Protected

Academic year: 2020

Share "Reduced beta 1 receptor messenger RNA abundance in the failing human heart"

Copied!
10
0
0

Loading.... (view fulltext now)

Full text

(1)

Reduced beta 1 receptor messenger RNA

abundance in the failing human heart.

M R Bristow, … , P E Ray, A M Feldman

J Clin Invest.

1993;

92(6)

:2737-2745.

https://doi.org/10.1172/JCI116891

.

Heart failure in humans is characterized by alterations in myocardial adrenergic signal

transduction, the most prominent of which is down-regulation of beta 1-adrenergic receptors.

We tested the hypothesis that down-regulation of beta 1-adrenergic receptors in the failing

human heart is related to decreased steady-state levels of beta 1 receptor mRNA. Due to

the extremely low abundance of beta 1 receptor mRNA, measurements were possible only

by quantitative polymerase chain reaction (QPCR) or by RNase protection methods.

Because the beta 1 receptor gene is intronless and beta 1 receptor mRNA abundance is

low, QPCR yielded genomic amplification in total RNA, and mRNA measurements had to

be performed in poly (A)(+)-enriched RNA. By QPCR the concentration of beta 1 receptor

mRNA varied from 0.34 to 7.8 x 10(7) molecules/microgram poly(A)(+)-enriched RNA, and

the assay was sensitive to 16.7 zeptomol. Using 100-mg aliquots of left ventricular

myocardium obtained from organ donors (nonfailing ventricles, n = 12) or heart transplant

recipients (failing ventricles, n = 13), the respective beta 1 mRNA levels measured by

QPCR were 4.2 +/- 0.7 x 10(7)/micrograms vs. 2.10 +/- 0.3 x 10(7)/micrograms (P = 0.006).

In these same nonfailing and failing left ventricles the respective beta 1-adrenergic receptor

densities were 67.9 +/- 6.9 fmol/mg vs. 29.6 +/- 3.5 fmol/mg (P = 0.0001). Decreased mRNA

abundance in the failing ventricles […]

Research Article

Find the latest version:

(2)

Reduced

flB

Receptor

Messenger RNA Abundance in the Failing Human Heart

MichaelR. Bristow, *WayneA. Minobe,*Mary V. Raynolds, * J. David Port, * RandyRasmussen,*Phillip E.Ray,'and Arthur M. Feldman*

*TempleHoyneBuell Laboratories, Division of Cardiology, University

of

ColoradoHealthSciences Center,Denver, Colorado

80262;

tDepartment

of Biology, University of Utah, Salt Lake City, Utah

84112;

andgThePeterBelferLaboratoryfor the Molecular Biology ofHeartFailure, The Johns HopkinsUniversitySchool ofMedicine, Baltimore, Maryland 21205

Abstract

Heartfailure in humans is characterized by alterations in myo-cardialadrenergic signal transduction, the most prominent of which is down-regulation of

ftl-adrenergic

receptors. We tested the

hypothesis

that

down-regulation of

fl1-adrenergic

receptors in thefailing human heart is related to decreased steady-state levelsof

61

receptor mRNA. Due to the extremely low abun-danceof

fih

receptormRNA, measurements were possible only

by

quantitative polymerase chain reaction (QPCR)

or by RNase

protection

methods. Because the

j8

receptor gene is intronless and

81

receptor mRNA abundance is low, QPCR yielded genomic

amplification

in total RNA, and mRNA mea-surementshadtobe

performed

in

poly(A)'-enriched

RNA.By

QPCR

the concentration

of

jI

receptor mRNA varied from

0.34

to

7.8

X107 molecules

/,g poly(A)'-enriched

RNA, and the assay was sensitive to

16.7

zeptomol.

Using

100-mg ali-quots of left ventricular

myocardium

obtained

from

organ do-nors(nonfailing ventricles, n=12) or heart transplant recipi-ents

(failing ventricles,

n=

13),

the

respective

jI

mRNA levels measured by QPCR were 4.2±0.7 X

107/,ug

vs. 2.10±0.3 X

107/i.tg

(P

=0.006). In these same nonfailing and failing left ventricles the respective

jBI-adrenergic

receptordensities were

67.9±6.9

fmol/mg

vs.

29.6±3.5

fmol/mg (P

=

0.0001).

De-creased mRNA abundance in the

failing ventricles

was

con-firmed

by RNase

protection

assays in totalRNA,

which

also

demonstrated

a

50% reduction

in

#1

message abundance. We conclude that

down-regulation of

01

receptor mRNA contrib-utesto

down-regulation

of

#I

adrenergic

receptors in the

failing

human heart.

(J. Clin.

Invest.

1993.

92:2737-2745.) Key

words:

,B-adrenergic

* heart

failure

-messenger RNA * polymer-ase

chain reaction

* receptor

Introduction

The

failing

human heart

is characterized by desensitization

to

adrenergic

stimulation (

1-5),

which

results in

attenuation of

the

ability

of

the hearttoincrease

contractility during

exercise

or

physiologic

stress. The

majority

of

this

subsensitivity

to

adrenergic

drive is

the result

of

a

selective

down-regulation

of the

myocardial

,B,-adrenergic

receptor

(2-5).

Inthe

failing

hu-Address reprint requeststo Dr. Michael R.Bristow, Temple Hoyne Buell Laboratories, Division ofCardiology, Universityof Colorado HealthSciencesCenter,Denver, CO 80262.

Receivedfor publication12November1992andinrevisedform21

July1993.

man heart ventricular myocardial fl-adrenergic receptors ap-pear tobe down-regulated in all subcellular fractions (4). Since there is nosignificant ,B receptor reserve in the human heart (6, 7),

#I

receptordensity is a major control point in the regulation ofcontractility (5). Less clear are the molecular alterations

responsible for

myocardial

jI

-adrenergic receptor down-regula-tion in thefailing human heart.

Inmodel systems the steady-state abundance of

12-adrener-gic receptor mRNA and receptor protein levels correlate closely (8, 9). Further,

studies

in cultured heart cells ( 10, 11 ) suggest that thesamerelationship between mRNA abundance and receptor

expression

may also

exist for

the

f31-adrenergic

receptorsubtype.

Accordingly,

wetested the hypothesis that, in the human heart, the steady-state abundance

of

/3,-adrenergic

receptor mRNAwould correlatewith j3, receptor expression at theprotein level. Because ofthe extremely low abundance of

,B

receptor mRNA, we measured

,3,

receptor mRNA levels by

quantitative

polymerase

chain reaction (QPCR)'

(12-14) or

by

RNase

protection (

1

5)

assays.

Using QPCR

with

poly(A)+-enrichedRNA asthe

starting

materialorRNaseprotection in total RNA, the data support the

premise

that

down-regulation

of

#,-adrenergic

receptors in

failing

human ventricular

myo-cardium is related

to

decreased

#3-adrenergic

receptor mRNA abundance.

Methods

Tissue procurement

Humanventricularmyocardiumwasobtainedfromsubjects undergo-ing heart or heart/lung transplant, and from organ donors whose heartswereunsuitable for donationowingtoblood typeorsize incom-patibility.15nonfailingleft ventriclesweretakenfrom14organ donors

and onesubject undergoing heart/lungtransplantation, with all 15 heartshaving normal left ventricular function determined by echocardi-ography. Failingleft ventricularmyocardiumwastakenfrom 13 sub-jects with end-stage left ventricular failure dueto idiopathicdilated cardiomyopathywho underwenthearttransplantation.Thesesubjects were notreceiving intravenous,B-adrenergic receptor agonists, phos-phodiesterase inhibitors, orantagonistsbeforetransplantation.Mean

age of thenonfailingcontrolswas28.8±5.1(SEM)yr,comparedwith 41.7±4.7 yr for thesubjects with heart failure (P=NS).Eightof the

nonfailing

controlswerefemale, andseven were male. Of thefailing subjects, 11weremale andtwowerefemale.

Allsubjects with end-stageheartfailurehad New York Heart

Asso-ciation classIVsymptoms beforetransplantation.Their

hemodynamic

data includedaleft ventricularejectionfraction of0.16±0.01,cardiac index of 2.01±0.23

liter/min/m2,

meanpulmonary

capillary

wedge pressure of 20.5±2.3 mmHg, mean pulmonary artery pressure of 30.5±2.6mmHg, andmeanrightatrial pressure of 6.6±2.3mmHg.

Tissuealiquotswereremovedfrom thestill-beatingheart immedi-ately uponexplantation,andeither immersedinliquid

nitrogen (see

1.Abbreviation usedinthis paper:QPCR, quantitativePCR.

J.Clin.Invest.

© The AmericanSocietyfor Clinical

Investigation,

Inc.

0021-9738/93/12/2737/09

$2.00

(3)

below)orplaced in ice-cold, oxygenated Tyrode's solutionasdescribed previously (2).

RNA

extraction

1-galiquotsof human left ventricular mid free wall were removedon

explantation, immediately immersed in liquid nitrogen, and stored at -70'C until used.Poly(A)+-enrichedmRNA wasextractedfrom 50-100-mg aliquots of human left ventricular myocardium using oligo(dT) cellulose

(Micro-FastTrack"

mRNA Isolation Kit Ver. 1.2,Invitrogen Corp., SanDiego, CA). Total RNA was extracted from separate 50-100-mg aliquots by the acid guanidinium thiocyanate phenol-chloroform method ( 16) using RNA Stat-60 (TelTest, Inc., Friendswood, TX), aspreviously described ( 13).

Quantitative

PCR

1,B

receptormRNA measurement. The basic technique for measuring mRNA abundance by QPCR in small aliquots of human heart has beenpreviously described ( 16). The ethanol precipitated pellet of ex-tractedpoly(A)+-enriched RNA (0.8-3.2Mg)wasdissolved in 16 Ml of diethyl pyrocarbonate-treated H20, and a 4-Ml aliquotwas directly measured by acapillaryspectrophotometer (model DU-64, Beckman Instruments, Inc., Fullerton,CA). ForRNAextracted from both non-failing andfailinghearts 200ng(1-4Ml)ofthe remaining 12 Ml ofRNA

was then added to the reverse transcriptase reaction, along with 0.5 pg ofasynthetic (84mer) cRNA "internal" standard ( 17). Thereverse

transcriptase reactionwascarriedoutaccordingtomanufacturer's in-structions (Promega Corp., Madison, WI), using a20-Mlreaction vol-ume, RNaseH-free Moloney murine leukemia virusreverse transcrip-tase, and randomprimers. The synthetic RNA (or cRNA) was gener-ated by an in vitro transcription reaction from synthetic DNA templates having a T7 RNA polymerase promoter sequence. The cRNA was DNAse (RNase free) treated, phenol/chloroform

ex-tracted, and ethanolprecipitated before resuspension and quantifica-tion by spectrophotometry. Theamountof cRNA addedtothe reverse transcription reaction was determined experimentally toultimately yieldcollinear amplification of the 131 receptor and internal standard

cDNAs.

Extensive characterizationofamplification of reverse transcribed 13, receptor mRNA resulted in selection ofthe

following optimal

condi-tions: 2% DMSO and 2 mMMgCl2 in the Taq polymerasebuffer,the useof RNaseHtodegrade theRNAstrand in theRNA:cDNAduplex afterreversetranscription, 50 pmol of each primer, and4Uof Taq polymerase (PromegaCorp.) per100-Mlreaction. Additionally, the re-action mixturewasdenaturedat94°Cfor 5 min, then cooledtothe annealingtemperature of55°C for 2min, andbroughttothe72°C extension temperature before Taq polymerasewasadded (18). Typi-callya4-Mlaliquot ofthereversetranscriptase/RNAse H-treated reac-tionwasused in the PCRreaction, withafinal volumeof 100M1.

Theprimers utilized in the 131 receptor PCR reaction spanned a 202-bp segment ofthe human131 cDNA sequence (+ 150 to +351 [17 ])

andincorporated restriction enzyme recognition sites at the 5' ends for

usein thecloning of this segment for other purposes. The sequences were: 5'.CCGAATTCCGAGCCGCTGTCTCAGCAGTGGACA * 3'

for the 5' primer and 5'- CCAAGCTTGGTGGCCCCGAACGGC-ACCACCAGCA-3'for the 3' primer. For subsequent quantification of PCR product yield after gel electrophoretic separation, an aliquot of

the3'primer was end-labeled with

[y-32P]ATP

using T4 polynucleo-tide kinase (Promega Corp.). All DNA oligonucleotides were pur-chasedfrom Operon Technologies, Alameda, CA. As described previ-ously ( 13),

10-,Ml

aliquots were removed from sequential amplification cycles (15through 33) and fractionated with molecular weight stan-dardsby agarose gel electrophoresis. Specific product bands were

lo-cated by ethidium bromide staining and ultraviolet irradiation, ex-cised, and quantified by Cerenkov counting.

With this technique, separate amplification curves for the unknown

cDNA and the internal standard cDNA were constructed. Since a knownamountofsyntheticRNAinternal standard is carried through the cDNA

synthesis

and

amplification,

the

original

13

receptor mRNA

level can be determined by extrapolation from the internal standard cDNA standardcurve. It is essential that thisextrapolation be per-formed during the exponential phase of amplification and that the curves for the unknown and internal standard are collinear, which occurred in allcasesbetweencycles 20 and 30. An RNA samplenot

subjectedto reversetranscription isrun as acontrol for contamination bygenomic DNA.

12 mRNA abundancewasmeasured in total RNA with linear

am-plificationoccurring in all cases between cycle numbers 20 and 30. The assay conditions for measuring 12 receptor mRNA abundance by QPCR have beenpreviouslydescribed (13, 19); 1 ggof total RNA

from bothfailing and nonfailing groups plus 30 pg of cRNAwasused in thereverse transcriptase reaction. Typicallya 1 glaliquot of the reversetranscriptase/RNaseHtreated reactionwasused inthe PCR reaction.

Measurementof

1,I

receptormRNAbyRNaseprotection.The hu-man

1,-adrenergic

receptor cDNA(17)wassubcloned intopBluescript

IIKS (Stratagene, Inc., La Jolla, CA) anddigestedwith either SmaIor

EagI. AntisenseRNAtranscribed from the T3 promoter of the SmaI digestedvectorcomprised- 450bpof the terminal end of the 3'

un-translatedregion(SmaI probe, probe I).Antisense RNA transcribed from the T3 promoter of the EagIdigestedvectorcontained - 400bp of the 3' end of thecoding regionandtheentire 3' untranslatedregion (- 700 bp) (EagI probe, probe II).

Antisenseriboprobesweretranscribed from theappropriately di-gested 131 receptor cDNAusingtheMaxiscribe kit(Ambion,Inc.,

Aus-tin, TX): The riboprobes were labeled by incorporation of [

a-32P]UTP800Ci/mM (New England Nuclear, Boston, MA) into the

RNAduringtranscriptionwith T3 DNA-dependentRNApolymerase. Approximately 1-2 X 105 cpm of32P-labeledantisense

1l3

RNAwas

used in each nucleaseprotectionassay.

Labeledriboprobeswerehybridizedinsolution with 2or5 yg of totalRNAextractedfromfailingornonfailingexplanted hearts for 18

hat44°C.Unhybridized single-strandedRNAwasdigested with

RNa-seA and RNaseT1. RNA-RNAhybridswereresolvedby electrophore-sis inan8%acrylamide denaturinggel and visualized by autoradiogra-phyat -80°C. The RPAII kit (Ambion, Inc.)wasusedforsolution hybridization and RNase digestion.

ProtectedRNAspecieswerequantified by densitometry.Inall ex-periments in vitro transcribed13,receptor sense strandRNAgenerated by thesamemethodfrom thesame vector astheantisenseriboprobes servedas apositivecontrol.Inaddition,in ordertoverifythespecificity of the 131 probes for

3,1

mRNA,thesyntheticfull-length12mRNA(20) wastranscribed and100pg hybridized with each ofthe two 131 antisense riboprobes.

Hybridization intensityfor protected RNA species of the appro-priate sizewasmeasured by densitometry ofautoradiograms developed for2-7d.Two orthreenonfailingcontrolsamples wererunoneach gel and the ODreading averages ofthese samples gave the 100%orcontrol value. Each samplefrom failing or nonfailing hearts was then refer-encedtothis 100% value, with results reportedaspercentageofcontrol.

13-Adrenergic

receptorquantification.

#1I

and

12

adrenergic receptors werequantified in a crude membrane fraction prepared from 5-g ali-quots of left ventricular free wall,using previously described methods (2).Briefly,the totalpopulation of

13I

plus

32

receptors wasmeasured by the nonselective radioligand

[1225l]iodocyanopindolol.

Maximum binding

(Bm..)

and the

['1251]iodocyanopindolol

dissociation constant (Kd)weredetermined by nonlinear least-squares computer modeling of thespecificbindingcurve(2).Fractions of

1,3

vs.

12

receptors in the membranepreparations were determined by computer modeling of competition curves using the highly selective

13,

antagonist

CGP-2071 2A(4).Multiplication ofthe totalBma,by the fraction of

13,

recep-tors obtained from CGP-20712A/[

'251]iodocyanopindolol

competi-tioncurvesthenyielded

3,1

receptordensity.

Protein concentrations were determined by the Peterson modifica-tion ofthemethodof Lowry (21 ). RNA concentration and purity was determined bymeasuring absorbance at 260 and 280 nM usingahighly sensitivecapillaryspectrophotometer (BeckmannInstrumentmodel DU-64).

(4)

A

B

C D E F G H I J K L M N O P Q R S T U V W

Figure 1. Assessmentof genomicDNAcontaminationlevels betweenpoly(A)'-enrichedRNA(H, I,J,K,P, Q, R, S) and totalRNA(L, M,N, 0, T, U, V, W) fractionssubjectedtoQPCR. 200ngofpoly(A)+-enrichedRNAand IAigof totalRNAextracted fromtwofailingandtwo

nonfailinghearts wereaddedtoduplicatereversetranscriptionreactionsdiffering onlyin whethertheycontainedreversetranscriptaseornot.

Subsequently, 10% of each of the totalRNAreactions and 20% of the

poly(A)'

reactionswereamplified by PCR. All thereversetranscription reactionswithtranscriptase(H I, J., K, L, M, N,0)showstrongamplificationofspecific 202-bp product (comparedwith thepositivesynthetic

f,

receptormRNAcontrol,(C)butof the reactions withouttranscriptase (P, Q,R,S, T, U, V, W)onlythe

poly(A)+-enriched

RNAreactions (P, Q, R, S) donotamplify the 202-bp product. (A)DNAmolecularweightmarkers. (B) PCRreagentblank.(C)Synthetic, full-length f3, re-ceptormRNA/positiveamplification control. (D, E, F, G) Synthetic

fA,

receptormRNA/noreversetranscriptasecontrols.

Statistical analysis

Comparison ofnumerical data betweentwo groups wasbyanunpaired

t-test.Correlationanalysiswasbysimple regression.Allstatistical

com-putationwasperformedontheStatView 512 +TM program

(Brain-power, Inc., Calabasas, CA). Statistical significance wastaken as P <0.05 inatwo-taileddistribution.

Results

QPCR

f3 receptor mRNA abundance. Shown in Fig. 1 is a 4% agarose

gel

stained with ethidium bromide

in

which

were run PCR products generated

from four

samples each

of

total and

poly(A)+-enriched

RNA, extracted

from

two

nonfailing

and two

failing

human

ventricles.

Inthe absence

of

reverse

tran-scriptase

[RT(-)

condition], genomic material of

the ex-pected

size for

the

Al

receptorPCR product

is

clearly present in the total RNAsamples

after

38 cycles. In contrast,

poly(A)+-enriched

RNA shows no

amplification

under

RT(-)

condi-tions.

Basedonthese resultsweused

poly

(A)+

-enriched

RNA asthe

starting material for QPCR

quantification of

f,

mRNA abundance.

Fig.

2 shows an

ethidium

stained

4% agarosegel in

which

were separated the contents

of six

PCR

reactions

using

poly(A)+-enriched

RNAasthe

starting material.

The

ampli-fied

cDNAs were made by reverse

transcribing 36-44

ng

of

poly(A)+-enriched

RNAextracted

from

six samples of

human

left ventricle

(designated A, B, C, D, E, and F) along

with

200-500

fg of

the

internal

standard cRNA. Each PCR

reaction

is represented by a set

of nine sequential

I0-yd

aliquots

which

wereremovedfrom thereaction tube at (left to right) cycles 14, 17, 19,

21,

24, 25, 27, 29, and 32. The PCR

amplification

product

for

Areceptor mRNA appears as a202-bp band,

while

the internal standard appears as an 84-bp band. The gel also includes "genomic controls": that is, poly(A)+-enriched RNA

from

the

six

samples (labeled,respectively, G-L) not subjected to reverse

transcription.

There was

progressive amplification of

the target mRNAand the internal standard cRNA in all six samples (A-F). However, there was no amplification of geno-micDNAin any sample (G-L). Identityof the 202-bp band was

confirmed

bydirect PCRsequencing (22).

Fig.

3 shows

amplification

curves

from four individual

sam-ples (two

failing

and twononfailing)using 1.9-11.4 fg of inter-nal standard cRNA per ng

of poly(A)+

RNA. It can be seen that there

is

collinear amplification

between20 and 30 cycles; curves wereaccepted for

analysis

only if

amplification

was lin-ear

for

at

least

six

cycles.

Inorder to comparethe

efficiency of

reversetranscription

of

the

internal

standard cRNA to that

of

the

f,

receptor mRNA, we measured by QPCR a

dilution series

of

cDNAs reverse

transcribed from

known, equal amounts ( 16.7 zepto-mol to 16.7 azepto-mol of each; or I04 to

107

molecules)

of

the cRNAand

full-length

f1 receptor mRNA. The latterwas gener-atedby in

vitro

transcription from

a

pBluescript

IIKS

phage-mid

expression

vector. An

amplification

curve wasconstructed

for

each

concentration of

the

series,

and

quantities of

mRNA vs.cRNA were calculatedfrom equivalent points in the

expo-nential

phase

of

amplification.

Asshown

in

Fig.

4, the

incorpo-ration

of

labeled

primers into

the PCR productsmade

from

the

internal

standard cRNAwas

essentially identical

tothose

for

the

full-length

f3 receptor mRNA. In orderto

determine

if the

(5)

stan-B

MW

-202

-84

C

D

G

H

I

J

K

L

Figure 2. Agarosegelscontainingthefractionatedcontentsof six separate PCR reactionsusing poly(A)+-enrichedRNAsubjectedtoreverse

transcriptionandPCR.Amplified

l,,

receptor mRNA appearsasa202-bpproduct, and the84-bpproductistheinternalstandard. MW, molec-ularweight markers.

10-,ul

aliquotswereremovedfrom the PCRreaction incycles 14, 17, 19, 21, 24, 25, 27, 29,and32, inlanes 1-9from left

toright. G-Larecycles 14,25,and 32for eachof sixsamplesinwhichreverse

transcriptase

wasomitted("genomic control").

dard,

the assay was

linear with

respecttoadded mRNA

from

measured

values

of 2.5

X

107

to

18

X

107

molecules/jig

poly(A)'

RNA.

Table I

gives

the

QPCR

quantification of

1l1

mRNA

in

poly(A)+-enriched samples from 13

failing

and 12

nonfailing

human hearts.

Compared

to

nonfailing

controls,

failing

hearts

had

a

50% lower abundance of

#I-adrenergic

receptor mRNA.

This

finding

was

similar

to

the 56%

lower

density of

1I

receptor observed

in the

failing

vs.

nonfailing

heart and

12

receptor mRNA

abundance.

132

receptor

mRNA abundance. 132

receptor

mRNA

abun-dance

wasalso measured

by

QPCR,

in total RNA

extracted

from

nonfailing

and

failing

human hearts(Table II). In con-trast to

13,

receptor

mRNA, collinear amplification of

reverse-transcribed

12

message

always

occurred in <

30 cycles.

Geno-mic

amplification

was not observed

for

the

132

mRNA assay, presumably because

of

the lower

PCR

cycle

requirements

(data notshown). As can be seen

in

Table

I,

there was no

difference

(P= NS)

in

132

receptor mRNA abundance or

12

receptor

den-sity

between

nonfailing

and

failing

hearts.

Relation between 13,

receptormRNA

abundance and

13,

or 2receptor

density. Fig.

5

gives

the

simple regression

relation

between

QPCR-measured

13 receptor mRNA abundance and

1,

or

12

receptor

density

inthe 25

(13

failing

and 12

nonfailing)

left ventricles.

There

is

a

significant direct relation between

131

mRNAand

3,B

receptor

density (top panel,

r =

0.52,

P=

0.008)

but no

relation

between 13 mRNA and

132

receptor

density

(bottom panel, r =0.07, P=NS).

RNase

protection

Nuclease

protection

assays were

also used

to measure 131 mRNA

abundance, in total

RNA

extracted

from explanted

failing and nonfailing hearts.

A

representative autoradiogram

from

an RNase

protection

assay

utilizing probe

Iin total RNA

is shown

in

Fig. 6.

In

Table

II are

given the

13,

mRNA

abun-dance

measurements

for probes

I

and

II,

andfor the average of both

probes

when both were used in

the

same

sample.

The abundance

of

13,

mRNA wasless

in

failing

than in

nonfailing

hearts,

confirming

the results

of

quantitative PCR. Using

the average data

for

both

probes,

13,

receptor mRNA was

decreased

by

50.2% and

13,

receptor

density

by 65.2%

in

the

same

hearts

(Table

II).

As shown

in

Fig.

7,

13,

mRNA measurement by RNase

protection

was

highly correlated

(r =

0.90,

P < .001) with QPCR measurements in the same samples, which

in-TableI.

1,1

and

132

Receptor mRNAAbundancesMeasured by QPCR and

13,

and 2ReceptorDensities in

Nonfailing

andFailingHumanLeft VentricularMyocardium

Group

(n) 1,3 mRNAabundance hi, receptor density fl2mRNA abundance 12receptor density

moleculesx

107/,sg

fmol/mg moleculesx107/;Lg fmol/mg

poly(A)+RNA totalRNA

Nonfailing

(12) 4.2±0.7 67.9±6.9 2.2±0.6 22.1±1.9

Failing

(13) 2.1±0.3* 29.6±3.5* 1.6±0.3 17.7±1.3

Valuesaregiven±SEM. *P <

0.01

vs.nonfailing controls.

2740 Bristowet al.

MW

E

F

MW

(6)

NON-FAILING

NON-FAILING

o BETAI

* CONSTRUCT o 0

-0 0

0 0

1o4Z

: 10'I

0

o*

0

S a

8

15 20 25

CYCLES

FAILING

10'

.

o BETAI * CONSTRUCT

0

. 0 104'

0 0 0 0

0 0 * @00

0

o 0

0

15 20 25

CYCLES

- 3

30 3 5

o

BETAl

* CONSTRUCT

0

.8

S a *

1 0

103

102

101

_

1 0

30 35

.

15 20 25

CYCLES

FAILING

o BETAI

* CONSTRUCT

0 0

0 0 0

0 S

8

15

20 25

CYCLES

Figure 3. PCR amplification ofreversetranscription products (cDNA) of

#,

receptormRNAfrom poly(A)+-enrichedRNA (., BETA 1)and

internal standard cRNA from the A,construct(-, CONSTRUCT).

cluded RNAextractedfrom fournonfailingand sixfailing

ven-tricles.

Toverifythespecificityof the antisenseprobes full-length sense human f2-adrenergic receptor mRNA was transcribed

andhybridized to bothantisense A, probes. ThetwoA3

anti-senseprobesusedwerehighly specificfor fl-adrenergic

recep-tormRNA,asneither the SmaI(probe I)northeEagI AI probe (probe II) protectedafragmentofl2mRNA ofthecorrectsize

(datanotshown).

Asthe

#I-adrenergic

receptorgeneisapparently intronless

(Fig. 1), protection ofgenomicDNA presentas acontaminant

in the RNApreparationscould beapotential problemasitwas with QPCR. Accordingly, RNA extracted froma nonfailing

heartwastreated with DNAse I beforehybridizationwith the

SmaIriboprobe. Bydensitometry the abundance offlI mRNA inDNAse I treatedtotalRNAwas nodifferent from the

abun-dance of fI mRNA in untreated total RNA from the same

sample (datanotshown).

Discussion

Using QPCRwe havepreviously reportedthat thefailing

hu-manheartexhibits marked abnormalities ofgeneexpression,

including de novo expression of atrial natriuretic peptide

mRNAanddecreasedlevels ofphospholambanmRNA( 13). Furthermore,thesechangesingeneexpressionarepotentially

reversiblewith restoration of normal cardiac function(23). Within the context of the altered receptor-G-protein-adenylyl cyclase signal transduction pathways in the failing heart, the molecular mechanisms responsibleforthe various

described abnormalities( 1-5)remainelusive.ThemRNA

lev-els for thef2adrenergicreceptor( 19)and the Gproteins,Gas

( 19) andGaj3 ( 19), are notdifferent intotal RNA extracted

from failing and nonfailinghuman ventricularmyocardium. This isnottotally unexpected,sincetherespectivegene

prod-uctsare not quantitativelyaltered in thefailinghuman heart

(5). Basedonpreviouswork inthe failinghuman heart(2, 3)

8

0 0 0 0

0 S

t

1o

a

I.

C)

1 0

10

10'4-a

103

102

10

0

30 35

0 0

S

0 S 0

30 3 5

101

-103-,

-2

(7)

-* BETA-1

y =3.8360+ 0.56342x RA2=0.965

.

0 INTSTND

y - 3.7934 +0.61796x RA2=0.975

-1 0

LOG amoles, INTERNAL STANDARD OR BETA-1 mRNA

Figure4. Simultaneousmeasurement of identical molar amountsof 3,1 receptor mRNA

(n, BETA-I)

and(31 cRNA internal standard

(standard

*,

INT

STND) by quantitative

PCR.

andincellculture modelsystems( 10, 11),oneelement ofthe

receptor-G-protein-adenylyl cyclase complexthat isastrong candidate for abnormal gene expression is the

#,(-adrenergic

receptor.

By Northern analysis or QPCR

(3I-adrenergic

receptor mRNA abundance measurements in total RNA extracted from human heart have thus far been unreliable, duetolow abundance of the G + C rich

(l3

receptor message (17) and genomic contaminationof PCRproducts. Basedonthe struc-turesofcloselyrelatedgenes(20, 24)and ourPCR data the

#,B-adrenergic

receptor

gene

likely

contains no

introns,

and

therefore PCRprimers couldnotbechosentodifferentiate the cDNA amplification signal from that ofgenomic DNA. The

needforhigh cyclenumbers(>35)indetectingthis

low-abun-dance messageexacerbatedthegenomicDNAcontamination problem. Consequently, it was necessary to use

poly(A)+-enriched RNAasthe starting material. Inusing these condi-tions, fewer than 32cycles ofPCR wererequiredtoproduce satisfactorylevels ofamplification. Furthermore, under these conditions, amplifications of non-reverse-transcribed

[RT(-)]

RNAproducednodetectablesignal, indicating the elimination

ofgenomic DNA contamination.

Inusing quantitative QPCR it is mandatory that collinear amplification be obtained with theinternal standard. This,in turn, requires that approximately equal amounts of internal standard and mRNAarepresent in the initialreactionmixture. Additionally, it is important that this collinear amplification

occur at relatively low cycle numbers (i.e., <35), to avoid artifacts causedby PCR. These criteria weresatisfied by the

above describedassayconditions.

Infailing ventricles, steady-state (3, receptormRNAlevels measuredby QPCRwere50%lowerthanin nonfailingcontrol hearts.Inthesesamefailing hearts receptordensitywas56%

lowerthan innonfailing controls. Moreover, when all 25

ven-tricles inwhich (3, receptormRNAabundancewasmeasured

wereconsidered, therewas adirect relation between (3dmRNA abundance and(,3 receptordensity, butnorelation between(,3

mRNAand(32receptordensity. The degree ofdown-regulation

ofmRNAandthedegree ofdown-regulation of (31-adrenergic

TableII.

(3I

ReceptormRNAAbundance MeasuredbyRNase Protection and

#,(-Adrenergic

Receptor Density

in Nonfailing and Failing Human Ventricular Myocardium

Probe I Probe II Both MO receptor

Group (Smal digest) (EagI digest) riboprobes density

%ofcontrol fmol/mg

Nonfailing 100.6±13.3 99.5±19.1 99.4±9.5 83.0±10.3 (n = 7) (n =4) (n =7) (n =7) Failing 52.3±9.0* 48.8±9.3* 49.7±6.6* 29.9±4.2*

(n = 6) (n =6) (n =6) (n =6)

Valuesaregiven±SEM.* P <.05vs.nonfailing.

receptors were

therefore

quite similar considering

the vast array

of factors which could

potentially influence

the

regula-tion of both

mRNA

and

proteins. These

factors include altered

transcriptional and translation

frequency,

mRNA

stability,

and

receptor

protein

turnover.

Also, the fact

that the

nonfailing

controls were obtained from organ donors who are invariably

subjected

tovery

high

levels of

endogenous sympathetic

drive due to brain

injury (25)

cannotbe

ignored.

Sincethe kinetics

of

mRNAturnover are

likely

tobe

faster than

receptor turn-over

(i.e.,

mRNA

half-life

vs.

protein half-life), it

may be that mRNA

abundance

was

already

beginning

to

decrease in the

nonfailing controls.

Decreased

(3,

receptor mRNA

in

failing left ventricles

was

confirmed by

RNase

protection

assay,

which showed

a

relative

decrease

in

message levels

by 50% in six

failing hearts. The

presence

of

Al,

receptor

genomic material contaminating

the total RNA in these assays

did

notpose a

problem, presumably

due to the

preference of

the

riboprobes for

RNA-RNA vs. RNA-DNA

hybridization (26). Of the

two

riboprobes,

the 3' UTR

probe

(SSmaI digest,

probe I)

gave

the best

hybridization

signal.

Both

probes

were

specific

for

(31

receptor

mRNA,

as

in

vitro transcribed

(32

mRNA did not cross-protect against RNase

degradation. The fact that in

two RNA

fractions

two

totally

independent

methods, QPCR and RNase protection,

yielded decreased

l,,

receptor mRNA abundance

measure-ments

in

failing

human heart provides strong evidence that

transcript

steady-state level

is

oneimportant determinant of

fl

receptor

regulation in

human ventricular myocardium.

Despite

the 50% decrease

in

flB

mRNA in

poly(A)+-enriched

ortotal RNAextracted

from

failing human hearts

(2

mRNA

abundance

in

total RNA was similar in failing and in

nonfailing ventricles. This is in

agreement with our previously reported data in totalRNA ( 19). Apparently because

(32

recep-tormRNA

abundance is

greater than

fl,

in the human heart,

QPCR

can be

performed in

total RNA with relatively low

(<

30) cycle numbers and genomic

contamination can be

avoided ( 19).

There aretwoprevious reports of QPCR measurements of

#I

receptor mRNA infailing human

ventricular

myocardium, one

reporting

an

increase

compared to nonfailing controls (27) andone

reporting

a

decrease

(28).

Inthe previous report

show-ing

adecrease (28), 38cycles of PCR were used on total RNA and noRT(-) controlcondition was reported. The report of an

increase

in

#I

mRNA in

failing ventricles

also used total RNA

(27).

Again,

the

combination of

low abundance and an

2742 Bristowetal.

C.)

0

-i

(8)

y = .043x + 1.125, R-squared: .302

y = -.027x + 3.673, R-squared: .006

8.

a)

Ca) 0

E 74 O

6 03

X

5.

E 1

| z 3 A ; m - - C1 a | Figure5. Simpleregression

13

° O3 °plots of

fB

receptormRNA

J 1 O3

(y-axis)

vs.

jl,

receptor

den-|3

m10 O |sity (toppanel)and

02

recep-0 . . . 2 tor

density

(bottom panel)

7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 in the 25left ventricles in

BETA2, FMOLUMG which ,S mRNAabundance

wasmeasured byQPCR.

1 2 3 4 5 6 7 8 9 10 II Figure 6. Arepresentative

autoradiogram ofanRNase

protectionassay.Total RNA extracted fromexplanted

hu-manheartswashybridized

withan[a32P]UTP-labeled antisensefl riboprobe(SmaI

450 bp fl,

450-bp

probe). Fragmentsprotected

from RNasedigestionwere

resolved bydenaturinggel

electrophoresisand visual-izedby autoradiogram.Lanes

1and2:SmaIantisensef3l

riboprobewithout (lane 1) and with(lane 2)RNasetreatment;lane 2 contains eight timestheamountof riboprobeaslanes or3-8. The smaller, faster migrating bands

presentinalllanesareprobablyself-protectedprobefragments.Asthe probe isveryG+ Crich(17), higher orderstructuresinthesingle

strandedantisenseprobearelikelytobepresent,evenunderdenaturingconditions. Thesestructureswillprotectportions ofthe probefrom RNasedigestion.Lanes 3, 4, and 5:synthetic f3ImRNA (200, 100,and 50 pg) hybridized withthe labeled,B riboprobe.Theprominentlower

bands in the standard lanesaremostlikely dueto prematureterminationof transcription ofthe syntheticsense

lB

mRNA. Lanes 6, 7, and 8:

Total RNAfrom three differentexplantednonfailinghuman heartshybridized withtheantisense

#I

riboprobe. Lanes 9,10,and 11: TotalRNA

from threedifferentexplantedfailinghumanhearts hybridized with the antisense

fl,

riboprobe. iB mRNA abundance is significantlylowerin

failinghumanheartscomparedtothe abundance in nonfailinghuman heartsasdeterminedby RNase protectionassay.

CD 0 0

C.)O 0E 7.

6

°0a) 6. 0

E 5

3.)

E O k 0 0

-2

OO

0)~~~~~

H 0

m

1.

OO0

0 20 40 60 80 100 120

BETA-1 RECEPTORDENSITY,fmol/mg

(9)

R-y = 14.013x + 30.974, R-squared: .817 130. 120 0 110 100 90. 80 * 70 * 60 40B*

30' .* .. . . 0 1 2 3 4 5

BETA-11 mRNA,moleculesx 10'7/mcg

Figure7.Correlation of

0,

mRNAabundancemeasur

poly(A)+-enrichedRNAvs.measurementbyRNasep

total RNA;fournonfailing and sixfailinghumanleft

intronless gene makes measurements ofAl rec

levelsby QPCR challenging,andunreliable in tol

high (> 30) cyclenumbers. Dependingonthe D tio in myocardium comprising the starting

"mRNA" abundance in total RNA could be fc creased ordecreased.

There are two previous reports of RNase p formedassolution hybridization with thehybri

filtration(29, 30). Althoughidenticalriboprobe

these two investigations (29, 30),onestudy in lated cardiomyopathy found an increase in "mRNA" (30) and the other in valvular hear

foundadecrease. Basedonthenumberofself-p

mentsdetected onacrylamide gelsin ourownI tion assays, it is obvious that the approl RNA:RNA hybrid needs to be separatedand i electrophoresis. Unlessit has been documented protected or a verydominantprotected produc

the overwhelming majority ofthe measured ra the solution assay, filtration isolation ofhybric

can lead tospuriousresults.Circumstancessuch

havecontributed tothedivergentresults inthese

studies(29, 30).

Although the starting material (left ventri( dium)usedfor mRNAandreceptormeasureme

cellular,themajorityofcellvolume,protein,an'

tricular myocardium is derived from cardiac

mammalian ventricles adrenergic receptors expressedinmyocardialcells(31 ),andtherefore

thevastmajorityof

A,

receptorproteinand mR

in thisinvestigation wasderived from cardiac

havepreviouslyshown thatthisis in factthecasc ergic receptors measured in crude membrane

tracted from humanventricles(2).

Insummary,f1 receptormRNAabundance

failing human heart than in nonfailing, contrc decreasecanfullyaccountfor theobserved dowr

fl1-adrenergicreceptorexpressioninfailing hearts

ies will be required todeterminethe molecular

alteration in geneexpression.

Acknowledgments

Wewish tothankDr.CarlT.Wittwerof theUniversiti

calSchoolforhelpful suggestionsonoptimal conditio 0

M. Benjamin Perryman for critical review of the manuscript, and Frank Stewart for manuscriptpreparation.

Thisworkwassupported byNational Institutes of Health(NIH)

grantsHL-13408-15andHL-48013-0IA2,awardedtoDr.Michael R. Bristow. Dr. Arthur M. Feldman was supported by NIH Program

Grant HL-39719 and The W. W. Smith Charitable Trust.

References

1.Bristow,M.R.,R.Ginsburg,W.Minobe,R.S.Cubicciotti,W. S.Sageman,

K.Lurie,M. E.Billingham, D.C.Harrison,andE. B.Stinson. 1982. Decreased catecholamine sensitivity and 3-adrenergic-receptor density in failinghuman

hearts. N.Engl.J.Med. 307:205-211.

2. Bristow,M. R., R.Ginsburg,M.Fowler, W.Minobe,R. Rasmussen, P. -edby QPCR in Zera, R.Menlove,P. Shah, and E.Stinson. 1986.,B and adrenergicreceptor

)rotectionin subpopulationsin normal andfailing humanventricular myocardium:coupling ventricles. of bothreceptorsubtypesto muscle contraction and selectiveA,receptor

down-regulationinheartfailure. Circ.Res.59:297-309.

3. Brodde, 0. R.,S. Schuler, R. Kretsch, M. Brinkmann, H.G. Borst, R.

,eptor mRNA Hetzer,J. Reidemeister, Warnecke, Regional

distribution of13-adrenoceptorsinthe human heart:coexistenceof function

/B,-talRNA using and,B2-adrenoceptors inbothatriaandventricles inseverecongestive

cardiomy-)NA/RNAra- opathy.J. Cardiovasc.Pharmacol. 8:1235-1242.

material,1 4.Bristow, M. R.,W. Minobe,R.Rasmussen, P.Larrabee, L.Skerl,J. W.

mato

be

in-

Klein,

F. L. Anderson, L. Murray, L.

Mestroni,

S. V. Karwande,

etal. 1992.

)undto be in- -Adrenergic neuroeffector abnormalities in the failing human heartare

pro-duced bylocal, rather thansystemic mechanisms. J.Clin. Invest.89:803-815.

Protection per- 5.Bristow, M. R., R. E. Hershberger, J. D. Port, E. M. Gilbert, A. Sandoval, R. Rasmussen, A. E. Cates,andA.M. Feldman. 1990. O-Adrenergicpathways in Ids isolatedby nonfailingand failinghuman ventricularmyocardium. Circulation. 82(Suppl.

swereusedin I):12-25.

idiopathic di- 6. Port, J. D., and M. R.Bristow. 1988.Lack of spare0-adrenergicreceptorsin

, receptor thehumanheart.FASEB J. 2:A602.

7. Brown,L., N. M.Deighton, S. Bals, W. Sohlmann, H. R.Zerkowski,M.C. I disease (30) Michel, and0. E. Brodde. 1992. Spare receptorsfor f3-adrenoceptor-mediated

Protectingfrag- positiveinotropiceffects of catecholamines inthe human heart. J. Cardiovasc.

RNaseprotec- Pharmacol. 19:222-232.

priately sized

gic8.

Hadcock,

J.R.,andC.C.Malbon. 1988.Down-regulationof beta-adrener-gicreceptors: agonist-induced reduction inreceptormRNA levels. Proc.Natl.

solated by gel Acad. Sci. USA. 85:5021-5025.

J that a single 9. Hadcock, J. R., H. Y. Wang, and C. C. Malbon. 1989.Agonist-induced

for destabilization of

f-adrenergic

receptormRNA:attenuationof

glucocorticoid-in-accounts or ducedup-regulationoffl-adrenergicreceptors.J.Biol.Chem.264:19928-19933.

tdioactivity in 10. Bristow, M. R.,C. Durham, J.Klein, R. Rasmussen, J. D. Port, R. R.

lized material Gesteland, W. H. Barry, and A. M. Feldman. 1991.Down-regulationof

f-adren-iasthesemay ergicreceptors andreceptor mRNA in heart cellschronically exposedto norepi-twoprevious nephrine.Clin.Res. 39:256A.(Abstr.)

two previous npI 1.Lazar-Wesley,E.,J. R.Hadcock,C. C. Malbon,G. Kunos, and E. J. Ishac.

1991. Tissue-specific regulationofalB1f1,andf2-adrenergicreceptormRNAsby

cular myocar- thyroid state in the rat.Endocrinology,. 129:1116-1118.

12.Wang, A.M., M. V. Doyle, and D. F. Mark.1989. Quantitationof mRNA ntswas multi- bythepolymerasechainreaction.Proc.Natl.Acad.Sci.USA.86:9717-9721.

JRNA inven- 13. Feldman, A. M., P. E. Ray, C. M.Silan, J.A.Mercer,W.Minobe, and

myocytes. In M.R. Bristow. 1991. Selective geneexpressioninfailinghumanheart.

Circula-are selectively tion.83:1866-1872.

14. Bouaboula, M., P. Legoux, B. Pessegue, B. Delpech, X. Dumont, M.

it islikely that Piechaczyk,P.Casellas, and D.Shire. Standardization ofmRNAtitration usinga

NA measured polymerase chain reactionmethodinvolvingco-amplification witha

multispeci-myocytes. We ficinternalcontrol. 1992.

J. Biol.

Chem. 267:21830-21838.

15. Lee, J. J.,and N.A.Costlow. 1987.Amoleculartitrationassaytomeasure

for:3-adren- transcriptprevalancelevels.Methods Enzymol. 152:633-648.

fractions ex- 16.Chomczynski,P., andN.Sacchi. 1987. Single-stepmethodofRNA

isola-tion by acid guanidinionthiocyanate-phenol-chloroform extraction. Anal.

Bio-is lower in the chem. 1622:156-159.

17.Frielle, T., S. Collins, K. W. Daniel, M. G. Caron, R. J. Lefkowitz, and

Al hearts. This B. K. Kobilka. 1987. Cloning ofthe cDNAfor the human beta I-adrenergic

i-regulationof receptor.Proc.Natl. Acad.Sci. USA. 84:7920-7924.

;.Further

18. Kawasaki, E.

S.

1990. Amplification of RNA. In PCR Protocols-A

GuidetoMethods andApplications.M. A.Innisand D. H.Gelfand,editors.

basis for this AcademicPress, Inc.,San Diego, CA.3-12.

19. Feldman,A. M., P. E. Ray, and M. R. Bristow. 1991. Expression of

a-subunits ofG proteins infailinghuman heart:areappraisalutilizing quantita-tive polymerase chain reaction.J.Mol. Cell. Cardiol.23:1355-1358.

20.Dixon,R.A.F., B. K. Kobilka, D.J.Strader,J. L.Benovic,H.G.

Dohl-man,T. Frielle,M.A.Bolanowski, C.D. Bennett, E. Rands, R.A.Diehl,etal. yofUtahMedi- 1986. Cloning of thegeneand cDNAformammalianfB-adrenergicreceptorand nsforPCR, Dr. homologywith rhodopsin.Nature(Lond.). 321:75-79.

Bristowetal.

OR

zf

z

(10)

21. Peterson,G.L. 1977. Asimplification of the protein assay methodof

Lowry etal. which ismoregenerally applicable. Anal. Biochem. 83:346-356. 22. Gyllensten, U. B., andH. A.Erlich. 1988. Generation of single-stranded

DNA by thepolymerase chain reactionandits applicationtodirectsequencing of the HLA-DQAlocus. Proc.Nati.Acad. Sci. USA.85:7652-7656.

23. Ladenson,P.W.,S.I.Sherman,K. L.Baughman,P.E.Ray, andA.M.

Feldman. 1992. Reversible alterations in myocardialgeneexpressionin ayoung

manwith dilated cardiomyopathy and hypothyroidism.Proc.Natl. Acad. Sci. USA.89:5251-5255.

24.Shimomura, H.,and A.Terada.1990. Primary structure of theratbeta-I adrenergic receptor gene. Nucleic AcidsRes. 18:4591.

25.Hamill,R.W.,P. D.Woolf,J. V.McDonald,L. A.Lee,and M.Kelly. 1987.Catecholaminespredictoutcomeintraumatic brain injury.Ann.Neurol. 21:438-443.

26. Sambrook, J.,E. F.Fritsch,and T. Maniatis. 1989. Molecular Cloning, a LaboratoryManual. 2nd edition. Cold Spring Harbor Press, Cold SpringHarbor, NY.

27.IhW-VahI,R., R. Marquetant, andR. H.Strasser. 1992. Subtype-selective regulation ofmRNAfor01-adrenergicreceptor in chronic heart failure.

Circula-tion.86(Suppl.I):I-767.(Abstr.)

28.Ungerer,M., M. Bohm, J. S. Elce, E. Erdmann, and M. J.Lohse. 1993.

Alteredexpression off-adrenergicreceptor kinaseand 3,1-adrenergicreceptorsin

thefailinghuman heart.Circulation.87:454-463.

29.Sylven, C.,M.Bronnegard, E. Jansson,P.Sotonyi, F.Liang-Xiong,F.

Waagstein, and A. Hjalmarsson. 1992. Increased

f3I

receptor expression at mRNAlevel in dilated cardiomyopathy. J.Am.Coll. Cardiol. 19:273A.(Abstr.)

30. Sylven, C.,P.Arner, L. Hellstrom, E. Jansson, P. Sotonyi, A. Somogyi, and M. Bronnegard. 1991. Left ventricular

f3,

andl2adrenoceptor mRNA

ex-pression in normal and volumeoverloaded human heart. Cardiovasc. Res.

25:737-741.

References

Related documents

Here again, we found three types of effects: a first one for which we observed an underestimation of the rates’ reaction function to macroeconomic announcements, when the effect of

The values PSNR and NC are increased as the number of iterations increased showing that genetic algorithms improves the image imperceptibility and robustness of

The definition of cloud computing provided by National Institute of Standards and Technology (NIST) says that: &#34;Cloud computing is a model for enabling

Споживання недостойних благ приносить відповідну антикорисність не лише його безпосереднім споживачам , а й особам , які не залучені до цього процесу , тобто

MPRA Paper No.. رﻳﺛﺄﺗ ﺔﻳﺑرﻌﻟا لودﻟا ﻰﻓ ﺔطﺳوﺗﻣﻟاو ةرﻳﻐﺻﻟا تﺎﻋﺎﻧﺻﻟا ﻰﻠﻋ ﻰﺑرﻌﻟا ﻰﻛرﻣﺟﻟا دﺎﺣﺗﻻا-. ﺔﺻﺻﺧﺗﻣﻟا ﺔﻳرودﻟا ﺔﻳﻧﺎﺳﻧإ

Making use of EI produced in 1997, for the oil industry operations in the Sound of Campeche [20] , together with 1995 meteorological information of the same region [12] to