Vol.

174,

February

No.

3,

14,

1991

BIOCHEMICAL

1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

ACTIVATION

OF ALPHA-MYOSIN

HEAVY CHAIN GENE EXPRESSION

1196-1203

BY CAMP IN

CULTURED FETAL RAT HEART MYOCYTES

*+

P. Cupta

plahesh

Radovan Departments

of

*

Medicine,+

The University January

, Alexandre

Stewart+

and

ZaF*+"

Organismal

and " The Pharmacological

Received



, !Jadhu Gupta

Biology

and Anatomy

6 Physiological

of Chicago,

Sciences,

Chicago

IL 60637

3, 1991

SrnRY: The effect of cA!fP on cardiac myosin heavy chain (WC) gene expression in primary cultures of IP-day-old fetal rat heart myocytes was investigated. When myocytes were treated with either 10 I,I.~ forskolin or 1 rn!: 8bromo-cPPiP for 4P h, the relative amount of the V1 to-V3 myosin isoform ratio increased 3-fold. The abundance of alpha-MHC mFMA was also increased 3-to-4fold in forskolin treated vs control cells. Powever, no appreciable change was observed in the level of beta-WC rnFbrA. Tn addition, a 707 increase in the transcription rate of the cardiac WC gene was observed by nuclear run-on assay following treatment of cells with 10 c(M forskolin for 12 h. These results demonstrate the preferential induction of alpha+PC mF)JA by CAMP which is, in part, mediated by transcriptional activation of the gene. 0 1991 Academic Press, Inc.

The that

contains

genes

heavy

to

encoded

isoforms,

designated

alpha-alpha

as by

and heterodimer.

activities

with

lowest various

ATPase to

the activity

follow patho-physiological

a

is

active

alpha these as VI,

alpha-beta

shown

(WC)

chain

ATPnse

the

referred

proteins of

'

myosin

and V3.

The

cardiac

the

known in

to

the

expression (3-7).

two

expressed to

differ and

highest,

form

three are

myosin composed

whereas,

V2

enzymatic

their

the

is

an

V3 isoform

cardiac

WC

genes

that

can

be

them

thyroid

has

the been

modified hormone

(vetropolitan Heart

The

in

of

Senior Pesearch Fellow of American Feart Association whom all correspondence should be addressed. cj Recipient of Junior Research Fellowship of American (Metropolitan Chicago). Abbreviation: WC, ?'yosin heavy chain.

WC

(1).

pattern Among

molecule

ventricles,

he

pairs

isoforms

developmental stimuli

of myosin

respectively,

myosin having

subunit

The V, and V3 isoforms

homodimers,

V1 isoform (2).

are

associate

These

defined

Tn

beta

genes

beta-beta

principal

site.

and V2,

the

Association

Chicago),

by has

tc

Vol.

174,

been

No.

3, 1991

found

to

be

upregulation

of

the

affect

WC

expression.

takes as

of

VI

so

pharmacological

Tn

To

byp.ass

by

treating

with

the

the

betn the

role present

mRP!P

in

analogue

as

WC

expression

either

we in

transdltction

ion

adenylate

of

of

has

system myosin

of

Fetal

intracellular activator

the

beer

sevcrp!

cardiac

cultures

not

(9).

nrrvous

effect

cyclase

expression

I’owcvrr,

the

raised

do

pathway

examinc,d

we

aginp,

Furthermore,

ventricles

expression. adrcnerFic

primarv

the

no

the

complex, with

?he

which level,

(7,s).

in

of

VI

hormone

drlring

the

Yet,

aspects to

\J3

thyroid

alter

redistrihut haup

important

seen

not

does

gent‘s

the

the

(5,7).

from

levels

does

that the

some

isoform

dctrrmining

isoforms

switch in

COMMUNICATIONS

in

myosin

surge

l’1

it

date

explain

virtually

study,

receptor-C-protein cells

cA?lP

atri.a,

suggest

important

V3

hormone

levels

regulates

to

isoform

the

the

hormone,

that

not

to

of

the

studies ‘qn

does

thyroid

thyroid far

of

myosin

prior

hormone in

than

identified

life

thyroid

isoforms

Other

and

the

circulating of

myosin

al pba

Blone

RESEARCH

studied

downreglllation

example,

downreFulation

BIOPHYSICAL

factor

hormone

the

AND

potent

the

fetal

manipulation

(4,lfl).

and

during

with

playinp

most

thyroid

as

correlate

two

the

For

place well

BIOCHEMICAL

of rat

might

be

isoforms cA,\‘P

heart cAI\“P forskol

on

?U’C

ceJ!s. lcve!s in

or

FI-brono-cAYP.

EXPERIMENTAL

PROCEDURES: rell culture-Primary cultllrrs of cardinc myocytps -_-__--were prepared as described hy Nag and Cheng (11). Cul turrs generally consisted of more than 90% myocytrs, as measured by immunocytofl\rorescence with anti-myosin antibody (5). The cells were tre;lted with difforrnr aqcnts Forskolin was prepared as 3OCP x stock in .~b~nlllte after 72 h of plating. ethanol, and 8-bromo-cAYP as 100 x stock in culture medium. Analysis of myosin isoforms-f:yosin isoforms were separated by ______ - ________. - _._.__.._-____-nondenatllring electrophoresis (2) and quantitated hy densirometric gel scanning of gels. ccl lular P?!P from ~~11 tured Tsolation and hvhridization .________. ---------c--_-_ -- ---__---- of _--- RNA-Tot*! CsCl gradient method of Chirgwin cardiac myocytes was isolated by the --et .31 ETorthern and slot blot analysrs were performed as described prfviorlsly (12). using synthetic olignnrlclcotidc probes complementary to the rlnioue 3’(13) a1 pbaand heta+PT mRMAs. Thes(, probes are nontranslated sequences of rat similar to those used earlier (14) except that alpha-PIJ’C probe was modified at both 5’ and 3’ ends to increase the signal to noise ratio. The specificity of these probes was analyzed hy !lsinp P?‘P prtaparp,j from thyroxine-treated adult rat heart whisk expresses alpha-WC mP>‘A (5,7,11) and soleus muscle, which expresses WC mPNA identical to that of cardiac beta+HC mFNA (5). Figure 1 shows the sequences and specific hyhridizat ion of alpha and beta oli,gonuclpotide probes to rat ventricular and soleils muscle PKA, respectively. Nuclear transcription assay-Pun-on transcription assay \$a s __----__ run-on --5----------_--; -------For the isolation performed essentially as described hy Ausubel et al. (13). P-40 lysis buffer containing IO mM of nuclei, cells were suspended in Nonidet 10 m?l NaCl, 3 mY FlgCl2, and n.5% PJonidet P-40, for 5 min at Tris, pH 7.4, 4oc. The nuclei were pelleted by centrifugation at 500 x g for 5 min at 4’r and gently resuspended in ice-cold buffer A (50 mE( Tris pH 8.0, 5 mPI rlgC12, 0.05 mPI ECTA, 2.5 mM DTT, and 207 glycerol), and an al.iquot was 0.15 Y KCl, counted after staining in 0.04% solution of trypan blue. To label the nascent PNA transcripts, the nuclei were incubated in 0.4 ml GTP, and CTP, and with 0.5 mCi of buffer A32supplemented with 1 m?l each of ATP, 3000 Ci/mmol) for 30 min at ;iO;alphaP]UTP (D u P ont New England Nuclear, . The reaction was termjnated hy addition of 0.6 ml of buffer B (0.5 M 1197

Vol.

174,

No.

3,

1991

BIOCHEMICAL

T3 c

AND

BIOPHYSICAL

RESEARCH

Soleus

T3

Soleus

t

t

4

COMMUNICATIONS

BMHC

aMHC

3’

5’ GTGGGATAGCAACAGCGAGGC

aMHC

GGTCTCAGGGCTTCACAGGC

PMHC

Figure 1. sequences and characterization of alphaand beta-MHC oligonucleotide probes. Total cellular FNA from rat soleus muscle (soleus) and thyroxinetreated (0.2 &g/100 g body weight intraperitoneal injection for 7 days) rat cardiac ventricular muscle (T3) were analyffd by Northern blot alpha-MHC analysis. Filter strips were hybridized with [ P] labeled Autoradiography was performed by (left) and beta-MHC (right) probes. overnight exposure of filters to XAR-5 film. Arrows denote the position of 285 and 185 ribosomal FNA bands. The sequences of the probes are given at the bottom of the figure.

50 nW MgCl2, 2 rnV CaC12, 10 rnF Tris, pII 7.4, and 40 ug/ml of RNase free DNase I) and incubation for 5 min at 30’C. Subsequently, nuclear extracts were digested for 30 min at 42’C with 200 ug OF proteinase K and l”/ SDS. Unincorporated isotopes were removed hy precipitation of FNA with 10% trichloroacetic acid and 60 mF’ sodium pyrophosphate. RNA was extracted with phenol/chloroform/isoamylalcohol, partjally hydrolyzed in 0.2 N NaOH and precipitated in ethanol. The PNA pellet was dissolved in TFC solution (10 mEf N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, pP 7.4, 10 mPf EpTA, n.2: SDS, and 0.3 ?q EJaCl) and hybridized at 65’C with 10 ug of linearized plasmids pA50 (a “housekeeping cDNA probe”), pRS (bluescript), and pPCWC (rat cardiac myosin heavy chain cDNA probe) immobilized on nitrocellulose filters. Fach filter was washed in an excess of 2 x SSC for 1 h at 65’C, followed by incubation of the filters with 10 ug of RNase A for 30 min at 37’C. Filters were washed once more with 2 x FSC at 77’C for 1 h, dried on Whatman 3mm paper, and exposed to X-ray film. NaCl,

RESULTS:

The

effect

cultures

of

IP-day-old

expression

of

cells

treated

(Fig.

2).

mRNA control

in

CAMP

of with

Northern

on

cardiac

fetal was

alpha+HC

amount

relative

of

VI

the

10

rat first

to

cells

treated

with

(Fig.

3a).

For

V3

myosin for

analysis IO

heart

revealed

UPI forskolin

expression

at

1198

An

that was

in the

also

in

increase

be the

3-fold

greater slot-blot

higher

in

control of

than

the The

level.

untreated

abundance

primary in

isoform

to

than

h.

studied

myosin

observed

was 48

the

evaluation,

quantitative

was

myocytes.

detected

uP1 forskolin blot

WC

alpha+EC that

analysis

in

the

was

Vol. 174, No. 3, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Probe:

aMHC

Ribosomal

w-4* control

0

-

-2,

-

-

-1,

-

.2*l*

IL

18S-FSK -

-0.5. (pg)

RNA

Ribosomal

RNA

RNA (pg) Figure

2. Changes

in

myosin

pattern

isoenzyme

treatment with 10 pm forskolin and forskolin-treated (FSK) electrophoresis. Densitometric gels at 565 nm at slit width

performed

by

linear

increase

conditions was

washed

indicated

that

not

due

cells

when

the to

with

1 mV

8-bromo-CAMP

the

medium

given

inhibitor to

in

cease

for

the

with it

which

the

3F,C),

was

28:

This 0.5 not

expression

of

mM

activity

contractile

1199

observed

alpha-WC for

probe mPMA

the

treatment

further

a

alpha-EWC cl?EJA

following was

in verifying

the

blotted

effect

blocked

thus

ribosomal

concentrations

h.

resulted

after

the

mRPJA was 48

along and

(Fig.

in

RNA

alpha+HC

8-bromo-CAMP was

phosphodiesterase

increase

variations

RNA

Furthermore, with

300X

in

total

signal

rehybridization

rise

cultured cardiomyoctyes due to Yyosin isoform from control (C) seuarated bv nondenaturine Y ael I were produced by scanning the

of

hybridization.

observed the

amounts

hybridization

excess off,

A similar

groups.

the

DNA

in

48h.

were profiles 0.05 mm.

of

increasing

in

of

probe

was

blotting

for cells

two of

potentiated

3-isobutyl-l-methylxanthine, by of

inclusion cells.

a of

25 mM I(+ However,

in no

Vol.

174,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

A

COMMUNICATIONS

B C

FSK

PA50PBS

-

pRCMHC

-

28S18S-

DRCMHC

03

0 C Figure

Figure

3. Effect of 10 PM forskolin treatment on alpha-MHC mRNA expression in celular RNA was prepared from cultured cardiomyocytes. Total cardiomyocytes 48 h after the addition of 10 /IM forskolin (FSK) or the A: 10 /tg RNA from two groups was diluent (control) to the medium. analyzed by Northern blot analysis using alpha-MAC oligonucleotide probe. R: different concentrations of RNA obtained from control and FSKtreated cells were blotted on nitrocellulose membrane by using slot blot minifold. Filter strip was hybridized with alpha-MHC oligonucleotide probe. After the alpha-MHC probe was washed off the same strip was The relative changes in MHC rehybridized with 285 ribosomal cDNA probe. mRNA and ribosomal RNA were analyzed by scanning the autoradiograms using a laser densitometer. C: standard curves for slot-blot assay of alpha-MHC mRNA and ribosomal RNA from control and 10 [tM forskolin-treated cells.

4. transcri tion control and )Relative rate of A: The specificity of the forskolin-treated cultured cardiomyocytes. pRCMHC probe to MHC mRNA was characterized by Northern blot analysis using ventricular RNA obtained from thyroxine-treated adult heart rat. B: for nuclear run-assay nuclei were prepared 12 h after addition of forskolin (C) to the cells. Nuclei were incubated in the (FSK) (lo PM) or diluent presence of 500 PCi lalpha-32P]HTP for 30 min. The [32P]PNA was isolated and hybridized to plasmid DNAs (10 pg per slot) containing pA50 (housekeeping cDNA), pRCMHC (myosin heavy chain cDNA) or PBS (plasmid bluescript without insert). Following autoradiography intensity of spots was determined by laser scanning. Background hybridization to pBS was substracted from values for pRCMHC probe, values are expressed as mean f S.E. of 4 different experiments.

appreciable the

change

was

CAMP-inducing Recause

transcription effects rate

the

observed

agents CAMP and

of of

04

FSK

CAMP gene

has the

on

were been rate

of

alpha+MC by

using

in

expression

used

(data

not

shown

to

mediate

degradation mFMA

the

the

result run-on

1200

beta-MHC

nRNP.

when

any

of

shown). changes

processes

might

nuclear

of

in

both

(15),

we

an

altered

from transcription

the

rate

examined

of

whether

transcription assay.

A

2.2

Kb

Vol.

174,

long

No.

rat

3, 1991

cardiac

meromyosin

in

41,

rats,

this

Recause

of

which

the

could

also

since

CAMP

treatment

level,

we

gene, equal

used

numbers

of with

gene”)

gene

obtained

uM

longer

(24

shown),

the

myosin

were from

inclusion the

Tn

by

the

effects

the

beta-MHC

the of

this

106

,

mP)JA

information

to

after

12

WC

18

(1 to RNA

treatment

in probe

polymerase

704

results

were

Jlowever,

when

shorter

(6

transcriptional

ug/ml)

of was

“housekeeping

h.

pPCMAC

In

equal

gene

(a

for

gene

alpha-amanitin

of

Similar for

an

groups which

WC

pP50

forskolin

on

h.

by

probe.

in

the

of

forskolin

of

different

those

unchanged.

with

pRCMJlC

the to

alpha-WC

produced

the

from

rate

the

cpm)

of

the

specificity

study,

of

in

h)

or

activity

the

transcription

completely II

(data

transcripts

removal

of

unrestored

(17).

normal,

Also,

in

hypertrophy lines

mentioned cardiac

of above, MHC

which

to

only evidence must genes.

the

for

gene

the of

that

the

CAMP

that the

1201

the

are

observation

level

This

is

alpha

and

cardiac of

in

alpha+HC

contrast

are

These

alphal-adrenergic of

the

artery beta-FIHC response

that

include

hypertrophy

return

of

beta-WC

by

alpha-WC

mPNA

remain

constriction-induced is

stimulated

and

recognized

a few

receptor-

cardiac

in

to

downregulates there

affected.

with

pulmonary of

signals Despite

is

changes

expression

suggest

in

associated

is

cultured

However,

gene

Regression

case

generate

the

VHC

while the

state

(5,9,14).

cardiac

(16).

handing

back

steady

upregulates

beta-MHC

hypertrophy

aortic

levels

the

of

unchanged.

manner one

treatment

the

mRNA

which

only of

that

increase

a reciprocal

which

cardiac

found

beta-WC

hormone,

genes in

rre

agents

levels

expression

stimulated

These

4 x

4,

hybridization

the

thyroid

preferential

cardiac

Fig.

effect

of

-

transcription

treated

specific

CAMP-inducing

examples

mRNA

in

with

a smaller

beta-WC

of

cells

essentially

cells

the

approximate

PNA

whereas

treated

alter

comparable

of

this

probe.

leaving

other

shown

bases.

Jrowever

transcription

treated

were

rate

was

indicating

myocytes

two

cells,

control

The

DISCUSSION: mRNA

As

in

inhibited

not

reslllts

intervals,

of (1

and

7100 transcripts,

gene.

jLP-labeled

of

thyroxine-treated

gene

an

light

analysis

transcripts.

not

obtain

rate

control

forskolin,

isolated

observed.

reaction

to

the

blot

approximately two gene

alpha-MHC

the

the

cells

h)

does

counts

transcription

after were

experiments

of

used.

of

of

Northern from

the

transcripts

from

were

band between beta-MHC

in

hybridization;

that

nuclei

was

increase

equal

10

than

single

In

COMMUNICATIONS

region

obtained

with

rate

nuclei

nuclei

higher

our

coding

probe.

RNA

a

RESEARCH

the

a

homology

32P-labeled

for

as

probe(pPCMH!C)

the

of

were

as

transcription

number experiments,

cells

this

BIOPHYSICAL

ventricular

of

in

hybridized

some

used

used

cross-hybridized

assess

we

was

degree

used the

To

, comprising

hybridized high

probe

regarding

(pRCMHC)

MFC,

we

probe

of

AND

EWC cDNA

portion

(Fig.

BIOCHEMICAL

the only

only

(18).

interventions by

one

alphc-MFC

of

the mRNA

Vol.

174,

No.

changes

in

myosin an

3, 1991

abundance

isoforms

also

the

of

or

by sympathectomy to

during

of

CAMP may also aging,

in

of

This

Vl

was

which

an

the

animal

(4,lO).

the

the

previous

observed

increase

in

ratio

studies

upon

Vl

with was

of alpha-MHC

this

catecholamines

in which

isoform

Responsiveness of

of

treatment

the

downregulation of

COMMUNICATIONS

CAMP, eventually

in

depletion

RESEARCH

confirms

isoform

explain

which

BIOPHYSICAL

influence

the

agonists,

expression

AND

be changed.

proportion

beta-adrenergic

seen

under

will

increased

prevented

BIOCHEMICAL

is

gene

well

that

is

documented

(19). The data increase

obtained

in

transcription was

of the

rapid,

yet

increased in

isogene

due

differential at

slowly

level

a concomitant cannot

and

of

be excluded. to

transcription

rate

hybridization

involvement of the

5'

a larger

in

of flanking

has

been

shown

it

transcription region

this

should

for

results the

to

be

since

probe

effect

of

the rat

to

been

beta-MHC

CAMP response cardiac

alpha-MHC

require

here, of

CAMP

pRCMHC was CAMP on the

underestimated

message. will

regulated

influence

the

have

the

presented

that

probe

of the

shown

the

actin

Although

be noted might

which

sarcomeric

MHC mRNA species, gene

of

transcription

(20).

been under

rate

disproportionate of

elsewhere from

observed

mFNA level,

A similar

has

the

altered

of MHC gene

transcription

mRNA stability

alpha-MHC of

genes

that an

alpha-MIX

extent. of

of

particular of

the

(5,21),

to

the rate

rate

MHC

indicate

due

the

the

However, one

in to

of transcription

modification

specific

cross

to

and

assays

part,

contrast

catecholamines

the

run-on in

The increase in

expression

largely

not

gene.

mRNA levels to

nuclear

mRNA is,

slight,

more

increase

from

alpha-MHC

The the

due

to

precise analysis

gene.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13

Nguhen, H.T., Gubits, R.M., Wydro, R.M., and Nadal-Ginard, B. (1982' Proc.Natl.Acad.Sci. IJSA 79:5230-5234. Hoh, J.F.Y., Yoeh, G.P.S., Thomas, M.A.H., and Higginbottom, L. (1979) FERS Let1 97:330-334. Whalen, R.G., Sell, S.M., Butler-Browne, G.S., Schwartz, R., Bouveret, P., ant Pinset-Harstrom, I. (1981) Nature 292:805-809. Dowell, R.T. (1976) Circ. Res. 39:683-689. Lompre, A.M., Nadal-Ginard, F., and Mahdavi, V. (1984) J.Biol.Chem. 259:6437 6446. Clark, W.A.,Jr., Chizzonite, R.A., Everett, A.W., Pabinowitz, M., and Zak, R (1982) J.Biol.Chem. 257:5449-5454. Chizzonite, R.A., and Zak, R. (1984) J.Riol.Chem. 259:12628-12632. Lompre,A.M., Mercadier, J.G., Wisnewsky, C., Bouveret, P., Pantaloni, D. D'Albis, and Schwartz, K. (1981) Dev.Biol. 84:286-291. Izumo, S., Nadal-Ginard, B., and Mahdavi, V. (1986) Science 231:597-600. Winegrad, S., McClellan, G, Tucker, M., and Lin, L-E. (1983) .J.Gen.Physiol. 81~749-765. Nag, A.C., and Cheng, M. (1984) Pi0chem.J. 221:21-26. Chirgwin, J.M., Przybyla, A.E., MacDonald, F.J., and Rutter, W.J. (1979) Biochemistry 18:5294-5299. Ausubel, F.M., Brent, P., Kinston, P.F., Moore, D.D., Seidman, J.G., Smith, 1202

Vol.

14 15

16 17 18 19 20 21

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

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Activation of alpha-myosin heavy chain gene expression by cAMP in cultured fetal rat heart myocytes.

The effect of cAMP on cardiac myosin heavy chain (MHC) gene expression in primary cultures of 18-day-old fetal rat heart myocytes was investigated. Wh...
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