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
RESEARCH
COMMUNICATIONS
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