The effect of epidermal growth factor on chronotropic response in cardiac cells in culture

Vol. 146, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 889-897

July 31,1987

THE EFFECT OF EPIDERMAL GROWTH FACTOR ON CHRONOTROPIC RESPONSE IN CARDIAC CELLS IN CULTURE Simon

W. Rabkin 1

University

"*,

Paul

Cardiovascular of British

Research Columbia,

20ncogen Received

June

23,

Sunga,

Inc.,

and

Sigrid

Laboratories, Vancouver,

Seattle

Myrdal

B.C.,

3

CANADA

WA

1987

SUMMARY: Cardiac chronotropic response to epidermal growth factor (EGF) was assessed in chick embryonic ventricular cell aggregates. EGF at a concentration of 10 ug/mL but not at 5 ug/mL produced a significant (~~0.05) increase in cardiac beating rate. This was evident within 10 min, reached a peak at about 15 min and remained at that level for 1.5 hr or the rest of the observation period. The effect of EGF on cardiac automaticity was reduced but not abolished at a lower temperature (22oC) that is known to decrease the affinity of the EGF receptor and reduce the internalization of EGF. Hypothermia did not change the maximum increase in heart rate response fromisoproterenol although it altered the pattern of the response. Beta adrenoreceptor blockade with metoprolol only slightly altered the response to EGF. These data indicate that EGF produces functional effects'on the heart that may be mediated through EGF receptor linked mechanisms. o 1987 Academic press, inc.

The recognition

of

receptor

for

cellular

responses

however,

have

received

beating

rate

cardiac (1)

this

and that

2Present CANADA

little

ion

concentrations the

address: V6H 3Nl.

Faculty

that

dependent

and reduced

of

EGF with

and of

the

requests

Medicine,

growth

has improved

factor

our

EGF alters for the

the

ion

we chose

cell

for 4500

is

fluxes

cell

highly

reprints Oak Street,

of

EGF, alter

across

membranes

their

rhythmicity

membrane the

(2,3).

effects

temperature temperature

should

of

EGF would

of

to examine

environmental

and the

effects

that

generation

myocardial

(EGF)

understanding

The cardiac

systems.

We hypothesized

mechanisms

blockade

correspondence

cell

across

possible

The association

epidermal

attention.

are

adrenoreceptor

whom all

of

cells

study

lTo

in a variety

of

surface

cardiac

To further

EGF.

on cell

on data

in

to

peptide

existence

based

on changes

beta

the

of

on the

response

dependent.

be addressed. Vancouver,

B.C ,

0006-291X/87 889

$1.50

Copyright 0 I987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 146, No. 2, 1987 Reduction

of

and also

study

this

to which

of

whether

temperature

decreases

temperature

BIOCHEMICALAND

EGF-receptor EGF affected

was influenced

it

decreases subsequent the

cell

EGF binding

to

its

internalization is

interaction.

BIOPHYSICALRESEARCH

exposed

(1,4).

provides

The purpose

cardiac

chronotropic

by beta

adrenoreceptor

cell

of

(rate)

this

response

blockade

or

surface Thus,

a useful

COMMUNICATIONS receptor

reduction

approach study

in

to the

was to

determine

and to determine different

whether

environmental

temperatures.

MATERIALS Cell

AND METHODS

Culture

Chick embryonic ventricular cells were cultured using the method of DeHaan as we have previously described (5). Briefly, white Leghorn eggs were incubated in an automatic incubator (March Rollex, California, USA) for 7 days at 37.8 degrees C and 87% humidity. Hearts were then isolated under sterile conditions from the 7 day chick embryo. Bllod and connective tissue were removed under a dissecting microscope in a solution of balanced salts (DMS8) with the following NaCl 116 mM, KC1 5.4 mM, NaH2P04*H20 0.8 mM, Na2HP04*7H20 1 mM composition: and dextrose 5.6. mM. Disaggregation was carried out by 5 minute digestions in 0.005% trypsin (Gibco Laboratories, Burlington, Ontario), 0.1% BSA and 1*10 7 Dornase units DNase per mL DMS8 (Worthington Biochemicals, Frederic, NJ, USA) at 37 degrees C. After three digestions, the digests were diluted 1:5 in culture medium and the cells centrifuged for three minutes at 1000 G in a clinical centrifuge (International Equipment Co., Needham Heights, Mass., USA). Cell aggregates were prepared as follows: Cells were collected and the number of cells/ml counted using a hemocytometer. Cells were diluted in medium in 25 mL Erlenmeyer flasks so that total volume was 3mL with approximately 3.0 x 10 6 cells/flask (Falcon, Becton Dickinson, Oxnard, CA, USA). Cells were maintained in medium 919A (20% M199, 73% DBSK buffer, 6% fetal bovine serum, penicillin at 100 units per mL, streptomycin at 10 ug/mL, plus fungizone 0.25 ug/mL). DBSK buffer had the following composition: NaCl 116 mM, NaH2P04*H2 1.0 mM, MgS04.7 H20 0.8mM,NaHP04*H2 1 mM, dextrose 5.6 mM, CaC12 1.8 mM, and NaHC03 26 mM. The flasks were placed on the platform of a variable speed Rotator (American Rotator, Can Lab) in the incubator at 60 RPM. After 40 to 72 hours, as needed, flasks were emptied into a 35 mm x 10 mm petri dish and swirled briefly to centre the aggregates which are allowed to stick to the bottom for about 30 min in the incubator. Beating rates were determined as outlined previously (5). A pair of petri dishes are placed in larger dishes with intake for 5% CO2 in air, bubbled through water on the stage of a Wilovertinverted micrgscope in a connected infants incubator. Constant temperature was maintained at 37 C by a heat lamp and tbermostat control with the temperature probe on the microscope stage. After 30 min equilibrium, a group of 5 or 6 aggregates/dish were selected and the contraction rate recorded. When the beating rate was consistent, a sham procedure was conducted. Two mL of medium was withdraen, placed in a petri dish within the incubator, mixed well and returned to the aggregate dish. When the beating rate was stable (constant), the drug was added to the withdrawn medium and returned to the aggregate dish in a similar manner to the sham procedure. Beating rates were recorded for each aggregate every 2 min for first 30 min, every 3 min for next 30 min, and every 5 min for the second hour. Drugs

and Chemicals

Epidermal (Lexington,

growth factor was obtained from Collaborative Missouri, USA). Isoproterenol was obtained 890

Research from Sigma

Inc. Chem ica ,l

Vol. 146,No.

co., St. Louis, Pharmaceuticals. Canada). Data

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

2, 1987 Missouri, Culture

USA. media

Metoprolol tartrate was obtained from

was a gift from Gibco (Burlington,

Astra Ontario,

Analysis

Beating

rates

per minute and percent change from baseline were calculated as the mean and one standard deviation. Hypothesis testing analysis of variance. The null hypothesis was rejected if the probability of a Type I error was less than 5% (i.e., ~~0.05).

and presented

used

RESULTS Cardiac

cell

experiment, hours dish of

aggregates

beating

(Figure did

rates

1).

not

affect

at

that

at

the

control.

after

the

its

attained

were

found

to

of

EGF at

beating in

an increase

for

that an hour

EGF concentration

Analysis

of

the

a constant

rate.

be stable

rate.

addition,

level

higher

at

The addition

10 mcgm/mL resulted

10 minutes

beat

the

for

The addition in

rate

its

peak

and a half.

change

in

the

conditions

periods

of

beating

reaches

the

concentration

in excess of

EGF at which

at

was evident

15 minutes

(p
greater

rate

beating

rate

than

compared

the to the

37Oc. 0

Control

0

EGF lOpg/ml

A

EGF 5pg/ml

0 20 SHAM

Figure

+

Drug

60

40

80

100

120

Time (minutes)

1. This shows the beating rate of myocardial cell aggregates in control cells that had not received EGF (O), in other cells that received EGF 5 ug/ml (A), and 10 ug/ml (0). The data is The aggregates were observed displayed as the mean -+ 1 S.D. for 120 min.

891

2

and remains

loo-

4

of

within

120

m-

the

a concentration

in

beating

of

5 mcgm/mL in the

The increase

was significantly

percent

Under

Vol. 146, No. 2, 1987

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

20 E ‘s t%

10

E s

0

$ El -10

6 ZR

-20

0

EGF 10pglml

A

EGF 5@g/ml

-30 20

I

/

I

I

I

40

60

60

100

120

Time (minutes) This 2. aggregates exposed

Figure

shows the percent change in beating rate of in controls, those exposed to EGF 5 ug/ml to EGF 10 ug/ml. The data is from Figure 1.

baseline

data

illustrates

of

with

rate

(Figure

2).

cells

period the

higher

minutes

that

However,

in

to

the

two-hour

when cells

were

was evident. of

almost the

the

prevented

of

period.

in heart

a 15% increase

in

over

rate

the After

decreased

EGF, 10 ug/mL, reduction rate

in with

beating about

but

22W

being

was not

decreased

a reduction

in

after

EGF antagonized

rate

produced

by 22oC. the

evident

rate

EGF, 10 ug/mL,

However,

Heart

EGF.

during

22OC.

892

to

slowing

at

temperatures,

3).

(~(0.05)

exposed

37OC.

lower

(Figure

a significant

22OC than

heart

15

temperature.

persistently rate

but

and a half.

of

not

rate

40 min,

rate

within

hour

with

continued

The beating. at

next

aggregates

gradually

period.

rate

by environmental

with

a two-hour

beating

temperature

cell

dependent

less

the

cell

populations

over

alter

lower

isolated

to 22oC.

(~~0.05)

the

the

baseline

was associated

Initially,

observation

to

the

in control

not

was affected

was time

Beating

exposed

reduction

rate

at

rate

EGF did

thereafter

temperature

rate

heart from

of

EGF produces

minimally

exposed

was significantly in cells

of

automaticity

in

of

deviation

EGF was reduced

beating

The decrease

rest

little

in environmental

decrease

stability

Low concentrations

increases

cardiac

Reduction

the

showing

concentration

The response

over

the

myocardial and those

rate

did

difference

not

increase and

To adjust between

for

Vol. 146, No. 2, 1987

BIOCHEMICALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS

20

=t 3 m"

10

E ;

0 El s -10

6

-20

0

Control

0

EGF~OM$TI

-30

T

2;

4'0

6b

8b

160

120

Time (minutes) Figure

control

cells

(Figure

4).

22oc.

and cells The effect

However,

Beating addition

3. This maintained and cells

the percent 22 C. Control had received

exposed of

increased

to

EGF was calculated

of

heart

an effect

significantly

isoproterenol

change in cardiac cells that had EGF at 10 ug/ml

EGF to increase

EGF had less

rate of

shows at that

at (~~0.05)

1°-'Mto

the

media

at

rate

the

beating rate in not received EGF (0) are compared.

both

temperatures

was evident

lower

temperature.

in this

preparation

(Figure

5).

At

cells (0)

37OC,

at

37oC and

after the

maximum

30

a 3Pc. A 22'C. -30

I

20

Figure

4. This compares control cells at Figure 2).

40

Tirnr(minutesI

the difference 22 C (data from

893

/ 80

in beating Figure 4)

100

rate between and 37 C (data

the

120

EGF and from

Vol. 146, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

40

30 is) = $

20

0”

E .E

10

s i%J 0 5 *

-10

0

20

40

60

SO

100

120

Time (minutes) Figure

increase the

5. This exposed

in heart

to

shows the percent isoproterenol

rate

was about

120 min observation.

line

within

the

decreased

so that

to produce

it

was back

of

isoproterenol.

response

to

EGF and isoproterenol

This

of

was a greater

To determine beta

adrenoreceptors,

used.

Metoprolol

of

about

produced mainly rate

reduction

slowing of

metoprolol

in EGF.

of

the in

initial

heart

rate

after

There

were

no

20 to

3 versus under

rate

rate

EGF.

beating

metoprolol significant

120 min

after 894

of

EGF at

a

22OC.

through

metoprolol

was

to a maximum Metoprolol

to

EGF that increase

not between

to these

was evident in

presence

was clearly

exposure

in the

conditions.

rate

5% in the

differences

gradually

5) shows

to

period.

base-

hypothermia

was mediated

The maximum

to about

but

even

Figure

agent

response

35% above

(20 min)

response

blocking

throughout

of

hypothermic

the

decreased

heart

15% was reduced

from

pattern

120 min observation the

cells

isoproterenol

was evident

initial

than

in

about

The effect

EGF on beating

20 min after

EGF of about

presence

EGF with

the

effect

to

of

rate

two agents

(~~0.05)

end of

the

adrenoreceptor

significantly

a slight

after

beta

addition

22OC (Figure

sustained effect

the

the

The marked the

the

of

at

increased

beating

rate

and was sustained

by 20 min.

in

in these

and more

45% near

during

baseline

Comparison

response

whether

to

rate the

reduction

presence

kind

after

ca&diac beahing 37 C and 22 C.

baseline

beating

few minutes

a time-dependent

different

35% above

At 22OC,

first

than e in lo- 9 M at

at

beating of

metoprol

evident

in

EGF alone

agents(Figure

01.

and 6).

Vol. 146, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

0 EGF lO&mL A

lo-%

Metoprolol

0 Metoprolol

2b

--

0

4b

I

10.’ + EGF lOW/mL

e'o

IdO

40

Tim~(minutes) Figure

6. This shows the aggregates exposed of EGF 10 ug/mL.

percent change to metoprolol

in cardiac beating 10m8M in the presence

rate

in

or

cell

absence

DISCUSSION The major

contribution

produce

definite

unclear

but

chick

heart

is

debated.

et

al

effects

its

potential

has

been

of

the

effects

of

on cardiac

The present EGF was not marked

to

study mediated

The generation well

as the

function.

growth

EGF was shown

role

The presence

of

found

EGF in mouse

heart

However,

in as well

transport,

(9)

cardiac

is

cells

cardiac

cells

while of

supports

from tissue Kasselberg

EGF on ion a role

chronotropic

as adrenergic

to

heart

EGF in

th;- effect

neurons

and specifically

in the

of mesenchymal

(6).

and cholinergic

ion

that Its

the

human heart.

also

found

through

that beta

in these

for

responses

because

and cholinergic

cells

the

increase

in

beating

adrenoreceptors. had a very

minor

rate

produced

Beta

blockade

that

effect

on the

heart

by produced rate

EGF.

The mechanism attractive

(7)

is

automaticity.

bradycardia

response

et al

function

importance

study

to influence

adrenergic

EGF in cardiac

present

on cardiac

no EGF in

(.I),

the

recognized

Roberts

(-8) found

transport

of

of

hypothesis of intact

the

effect is

cardiac heart

that

of

EGF on cardiac

it

is

electrical is

dependent

operative activity on Na+,

rate

is

through

changes

in cultured CaZ+,

unknown

chick

but in ion hearts

and K+ currents.

an flux. as Each

Vol. 146, No. 2, 1987 of

these

may be operative

calcium

entry

into

increase

heart

a variety

of

mechanism

for

rate

is

which

is

rate cells the

by increasing

of

process. is

e.g.,

the

of

the

of

designed

temperatures

fusion

but

temperature.

smaller

than of cell

automaticity

at

these

the

half

saturated it

is

another

possible beating (13) Na

be due to activation

of

cardiac

is

rate

at

at

the

(17).

crucial

the

A physiologic

by producing

partial

The increase

This

in 896

effect the

effect

of

effect

rate

to

EGF at

is,

thus, study

to a

22oC was much

EGF by hypothermia

depolarization

then after

EGF

37 degrees

response

reduces

rate

of

different

temperature

heart

antagonized

The present

EGF at

heart

of

is

temperatures

effects.

the

EGF (1).

EGF receptor,

compared

of

the

concentration

e.g.,

effects

in

the

greater

lower

in of

of

(4).

at

antagonism

Reduction

step

internalization

degrees

maximum

lysosomes

first

affinity

to affect

in endo-

EGF within

antagonistic

ability

complexes

bodies,

37 degrees

the

the

clustering

multi-vesicular

the

response

the

with

a tenfold

zero

in a stepwise

receptors, of

the

in the

examine

We found

explanation.

of

at

potential

its

to occur

on phosphatases,

increased

these

(18,19).

to

vesicles

shift

chronotropic

cultures

in

EGF may increase

prevent

temperatures

370C.

urn entry

EGF increases

internalization

temperature

than

rather

calci

in culture,

(14).

EGF to surface

the of

to precisely

reduced

in

surface,

in

is

because

rate

of

receptors

on cardiac

interest

part

binding

surface

low

cell

been considered

EGF to

of

will

Na concentrations

thought

degradation

degrees

is

in

calcium

increase

myocytes

Thus,

EGF have

a temperature-dependent

The effect

only

types

and the

on phosphorylation

of

of

is

to

Na+ entry

the

lysosomes,

receptor

extracellular

extracellular

into

Increases

heart.

as well.

and the

effect

was not

Na entry

on the

EGF at zero

by the

of

the

(15,16).

Reductions

There

to

mechanism

its

found

In cardiac

cell

involves

presumably

EGF.

different

effects

vesicles

Binding

of

RESEARCH COMMUNICATIONS

EGF on the

by elevating

Alteration

into

EGF: receptor

of

(11,lZ).

Na entry

The cellular

cytic

produced

proportionate

exchange

that

effects

EGF has been

effect

in several

fashion

cell

AND BIOPHYSICAL

(10).

translated

a Na+/H+

in the

the

directly

uptake

of

BIOCHEMICAL

is

heart depression

isoproterenol

is

C. of

Vol.

146,

No.

also

partially

effect

of

2, 1987

prevented

of

thermia

on the

is

response

on cardiac although

prevented

was different.

This

with

cells

to the

under

binding

BIOPHYSICAL

EGF is

not

hypothesis

studies

suggests

simply

that

less

even the

cells

are

though

effect

to

normothermic required

maximum the

of

hypo-

antagonism.

EGF binding

than

COMMUNICATIONS

EGF, the

a physiologic

rather

in cardiac

to

by hypothermia

of

hypothermic

RESEARCH

In contrast

was not

response

consistent

AND

by hypothermia.

isoproterenol

pattern

It

BIOCHEMICAL

to

its

receptor

conditions come to a definite

conclusion. In summary, in cardiac mediated in cardiac

the

cells by beta cells

present namely

study

an increase

adrenergic is

found

supported

that in

EGF produces

chronotropic

receptors.

functional

response.

An EGF receptor-mediated

by a blunting

of

the

heart

rate

changes This

was not

effect response

to

EGF hy hypothermia. REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 79.

Carpenter, G. and Cohen, S. (1979) Ann. Rev. Biochem. 48, 193-216. In Developmental Sperekalis, N., Shigenbou, K., and McLean, M.J. (1975) Edited by Lieberman, M. and Physiological Correlates of Cardiac Muscle. Raven Press, NY. and Sano, T. Carmeliet, E., and Vereecke, J. (1979) In Handbook of Physiology, Section 2, Sot Bethesda, Maryland USA 269-335. Ed. Berne, R.M. Am Physiol P. (1982) J. Biol. Chem. 257, 3053-3060. King, A.C., and Cuatrecasas, Myrdal, S.E., and De. Haan, R.L. (1983) J. Cell. Physiol. 117, 319-325. Balk, S.D., Riley, T.M., Gunther, H.S., and Morisi, A. (1985) Proc. Natl. Acad. Sci. USA 82, 5781-5. Roberts, A.B., Anzone, M.A., Lamb, L.C., Smith, J.M., and Sporm, M.B. (1981) Proc Nat1 Acad Sci USA 78, 5339-43. Kasselberg, A.G., Orth, D.N., Gray, M.E., and Stahlman, M.T. (1985) J. Histochem Cytochem. 33, 315-322. Fukada, K. (1980) Nature 287, 553-5. Goshima, K, (1975) In Developmental and Physiological Correlates of Cardiac Ed. M. Luberman and T. Sono. Raven Press, NY, 197-208. muscle. and Cohen, S. (1981) Biochemistry 20, 6280-6286. Sawyer, S.T., Moolenaar, W.H., Tertoolen, L.G.J., and de Laat, S.W. (1984) J. Biol. Chem. 259, 8066-8069. McCall, D. (1976) J. Gen Physiol 68, 537-549. Ellis, D. (1977) J. Physiol. 273, 211-240. and Rosengurt, E. (1982) Proc. Nat1 Acad Sci (USA) 79,7783-7787. Smith, J.B., Rothenberg, P., Glasser, L., Schlesinger, P., and Cassel, D. (1983) J. Biol. Chem. 258, 4883-4889. A.M. (1981) Ann NY Acad Carpenter, G., Stoscheck, C.M., and Soderquist, Sci 397, 11-17. I.S. (1972) J. Molec & Cellular Cardiol. 4, 453-463. Boder, G.B., and Johnson, D. (1967) Am. H. Physiol 213, 719-724. Sperekalis, N., and Lehmkuhl, 897