Effects of antinomycin D on fibrillation activity in denervated skeletal muscles of the rat

Life Sciences Vol. 13, pp .176.3-1770, 1973 . Printed in Great Britain

EFFECTS

OF ACTINOMYCIN

D ON

FIBRILLATION ACTIVITY

SKELETAL MUSCLES Salomon

Muchntk,

Pergamon Press

Adolfo C .

IN

DENERVATED

OF THE RAT Ruarte

and Basilto A .Kotstas

Instituto de Investigaciones Médicas, Universtdad de Buenos Aires Hospital TornG, Donato Alvarez 3000, Buenos Aires, Argentina and Centro de Investigaciones Neurolbgicas, Hospital de Nttlos, Sala XVIII, Galio 1330, Buenos Aires, Argentina .

(Received in final form 19 October 1973) SUMMARY The affects of actinomyctn D do fibrillation activity, acetylcholine sensitivity and resting membrane potential of Actinomyctn D denervated muscles of the rat was studied . (0 .7 mg/kg I .V .) administered 1 day after denervatton delays the appearance of fibrillation for approximately 3 days . If this drug is given 5-7 days after denervatton, it is also capable of blocking the already established ftbrillation but falls to suppress extraJuncttonal choltnergic receptors and to reverse the fall to resting potential . The mechanical responses of denervated muscles are unaffected by actinomyctn D . These results suggest that in fibrillation a genetic induction of newly formed RNA and protein is involved . It is also suggested that these molecules probably have a more rapid turnover than those required for the formation of extrasynaptic receptors in denervated muscle . The purpose of this actinomyctn The use

is the study of the effect

D on ftbrillation activity

of this

vestigation

paper been

drug has

recently

the XVII Clintca

results

reported

on

Mar del

in

already contributed

skeletal

Plate,

muscle

(1,2) .

in this paper were presented

meeting of the Soctedad Argentina de to

to the

Introduced

concerning the molecular basis of the

regulatory influence of motor nerves Some of the

Tn denervated muscles .

of denervatton phenomena and has

interesting suggestions

of

in

Investtgact5n

Argentina on November 1972 (3) .

MATERIAL AND METHODS Experiments were done on Wtstar rats weighing Denervation was thigh

100-180 gr .

performed by cutting the sciatic nerve at

under ether anesthesia .

ActtnomycTn D

1783

the

(Lyovac-Cosmegen,

1784

Actinomycin D and Fibrillation Activity

Merck,

Sharp b Dohme) was

injected Unless

diluted

into a veto of the

otherwise

separated by

tall

Indicated,

12

in

1

ml

Vol. 13, No. 12

of distilled water and

under light ether anesthesia .

the drug was given

to two equal doses Fibrillation was electromyographically

hours .

studied by means of thin coaxial needle electrodes connected to a 5103N Tektronix Oscilloscope through a P9 AC Grass Preamplifier . Photographs were taken with a 14A30 Disa 3-Channel Electromyograph .

"In vitro" studies been mounted medium

(4)

in a

kept

were done

Lucite

at

on

Isolated muscles

chamber filled with

pH 7 .2

and

the following composition :

20-24°C .

(mM)

C1

Na +

that

had

an oxygenated

The bathing solution

162,

K + 5,

Ca ++ 2,

had

Mg ++

1,

148, HC0 3 24, H 2 P0 4 1 and glucose 11 . Intracellular recordings were obtained by means of glass-microelectrodes and standard electrophysiological techniques . Iontophoretlc application of acetylcholine (ACh) was performed according to the procedure described by Axelsson and Thesleff to ACh

is

expressed

Isometric mechanical studied "in a Model

to Units a

Polygraph .

FT03

Grass

Section of the muscle was length

1

(5) .

mV/10 -9

Sensitivity Coulombs) .

stimulation were

Strain

Gauge connected

to

stimulation consisted of

field electrodes

long axis of the muscle .

resting

=

Electrical

square pulses delivered through to the

Unit

responses to electrical

vitro" using

7 Grass

(1

Tension

Is

placed

expressed

parallel

in g/mm 2 .

estimated dividing the weight by

the

(6) . RESULTS

1 .

Appearance of fibrtllary activity

onset

of

fibrillation was

in untreated rats .

investigated

frequency of the activity was

to

Oscilloscope . foci were all

the

the

fourth

22) .

On

rats with On

day, the

(50 msec duration)

Forty-eight hours

found .

that

in the screen of a

in

The

ten

Storage

few active

fibrillation was detected

a frequency of 94/sec ±

fifth day,

appeared

after denervation,

the third day,

the frequency was

point (358/sec ± 34) .

The rats .

estimated by counting the fibrilla-

tion potentials and positive sharp waves successive sweeps

10 denervated

almost

13

(mean ± S .E .) .

twice as high

fibrillation was

very

in

On

(174/sec

Intense at

every

Actinomycin D and Fibrillation Activity

Vol . 13, No. 12

2.

Effects of early

examine

infections of actinomycin D .

the effects of early

appearance of

ftbrtllation,

later .

It was assumed that

influence

is sttil

maintained

(7) .

In

by

in

This assumption

that neuromuscular transmission does

immediately after the sectYon of the nerve and some period of time

received

injection coincided approxi-

the baglnning of denervatlon .

the fact

D on the

rats were denervated and

these experiments the time of the based on

In order to

infections of actinomycin 10

0 .7 mg/kg of the drug 24 hours mately with

1785

its peripheral

these rats,

is

not

fall

"trophic"

end for a

short

stimulation of the severed

aciattc nerve no longer produces any contraction of the denervated muscles after

36 hours .

Denervated muscles examined on On

from these early

the fourth and

thn sixth day,

infected

rats were fibrillation .

fifth day and showed no

fibrillation was present

all

in

the muscles .

Thus development of fibrillation was delayed 3 days Jection 3. also

of actinomycin

Effects

spontaneous

D on

tibrtllating muscles .

see whether acttnomyctn

in denervated activity .

muscles that

Three rats

D was able

We ware to suppress

already exhibited

as

frequency of discharge was

reduced for two days .

tected

(Fig .

markedly

1B) .

and

a

but

this

Using a higher dose

inhibition was

found

In

dose was poorly tolerated

These results to delay the onset

of fibrillation

4.

change when

Imm edia te effects

it

is

In a

1A,

days the In-

second series,

(0 .7 mg/kg), (Fig .

rats

1C) .

A

a complete longer

treated with 0 .8 mg/kg

by the animals .

show that actinomycin

the section of the motor nerve but nervatlon

in Fig .

similar blocking effect was de-

blockage of fibrillation was obtained period of

shown

resumed on the third day .

0 .6 mg/kg was administered

intense

received 0 .5 mg/kg five

after the section of the nerve and, tense activity

ln-

D.

of acttnomyctn

interested to

ftbrtllation

by the

D

is not only able

(f administered early after it also

inhibits this

de

already established .

of actinomycin

D on ftbrtllation_ .

series of experiments was performed to discard an

This

immediate

effect of actinomycin D on fibrillation, bearing In mind that it takes 12 hours for the inhibition of muscle RNA synthesis to be fully developed after a single

infection

of the drug

(2) .

1786

Acünomycin D and Fibrillation Activity

A

B

Vol. 13, No. 12

C

Coafml

24

~^~W^

`~+~.,~

L

r

FIG .

1

Effects of actinomycin D on fibrillatiog muscles . Electromyographic records of denervated muscles from rats treated five days following denervation with various doses of the drug . Thus, A = 0 .5 mg/kg, S = 0 .6 mg/kg and C = 0 .7 mg/kg . Each column illustrates consecutive observations made on the same animal . Numbers on the left indicate hours after the injection of actinomycin D . Ttme calibration, 50 msec ; Voltage calibration, 50 uV . Six rats were fifth

injected with a single dose of 0 .7 mg/kg on

day after

Spontaneous

denervation

and completely disappeared 5"

Effects

and observed

activity persisted 6

for

hours

12,

24 hours

the

24 and 30 hours after

the

later .

injection

later .

on muscle excitability .

At

this

point,

it was

con-

sidered a matter of major concern to demonstrate that denervated muscles pite out

the

from treated

rats kept

and the mechanical

tion were studied The values follows :

their excitability unaltered des-

absence of fibrillation .

twitch

Soleus

responses to external

on the seventh day

found

muscles were dissected electrical

in five denervated control

tension,

3 .8 ± 0 .3

stimula-

following denervation .

g/mm 2 ,

muscles were as

tetanus tension,

15 .8 ±

Four rats were 3 " 5 g/mm 2 and fusion frequency, 15 .8 ± 5 c/sec . injected with 0 .7 mg/kg of actinomycin D on the fifth day and the denervated muscles significant

were

alteration

examined in

under the same

their mechanical

conditions .

output

No

became apparent :

Actinomycin D and Fibrillation Activity

Vol. 19, No . 12

twitch

tension,

mm 2 and

fusion

4 .6 ± 0 .4 g/mm 2 ,

response of both in

control

eration

is

not

to

strongly

affected

Effects on

known that two

and

treated muscles are suggest

(MPO)

(8,9) . The effects

(RSP) on

and

in

Fibrillation

10 fibers

and MPO

of three treated (30 fibers) .

muscles .

the synaptic area

all

animals

potential

oscillations"

under

A total

of them .

had been

Three

They were exam"in vtvo"

of 26 fibers

potentials were found

In

Is well

were analized as follows . control

in

12 fibers

In denervated soleus detected

injected with

0 .7 mg/kg

after the section of the nerve and observed

later .

7"

Effects of actinomycin D on

the

and

extr~uncttonal

to acet

sensitivi ty

experiments were performed mals were used as actinomyctn Two days

D

determined

by

lc~holtne .

the denervated soleus were

in 29

Similarly,

control

fibers was

fibers was

mV) .

104 ±

In

both

(52 .3

cases,

potential" .

86 ± 6

Units

all

The sensi-

(mean ± S .E .)

10 Units .

difference was observed

of control

(56 .9 ± 1 .1

"ACh

examined

acetylcholine was

No difference was

and treated muscles .

no marked

resting potential

ani-

denervation

removed and

sensitivity to

iontophoretic application .

treated

These

Three untreated

seventh day after

responded with a typical

tivity found 20

six rats .

on the

ExtraJunctional

found between control the fibers

in

resting membrane po t entia l

controls and the other three were given

(0 .7 mg/kg)

later,

"in vitro" .

muscles

gen-

called "rhythmic

rats, no spontaneous activity was

These

on the fourth day

in

It

resting

have been

six days after denervation .

were studied .

two days

They

such events

tned with microelectrodes

in

potential

usually accompanied by

"membrane potential

soleus were used as

conditions

are

low voltage oscillations of the

denervated muscle fibers .

denervated

that action

in treated muscles .

fibrillation potentials

types of

potentials"

and

Typical

illustrated

intracellularly recorded events .

small

RSP

17 .6 ± 3 " 7 g/

(mean ± S .E .) .

Figure 2 . These results

6.

tetanus tension,

frequency 20 ± 6 c/sec

1787

0 .96 mV)

between

and treated

the

Actinomycin D aad Fibrillation Activity

1788

Vol . 13, No. 12

CONTROL

~IL~I!!LI~~L~I

~~~

TREATED

e

d

f

FIG .

2

Effects of actinomycin D on the mechanical responses of denervated soleus muscles recorded "in vitro" 7 days after denervation . Control : untreated rat, muscle weight 100 mg, resting length 19 mm . Treated : 0 .7 mg/kg of actinomycin D was given 5 days after denervation, muscle weight 85 mg, resting length 20 mm . In a,b,d and e, isometric twitch responses to repetitive stimulation at 1 c/s are shown . In c and f, tetanic contractions to Time calibration, 15 c/s (c) and 27 c/s (f) are illustrated . a and d, 6 sec ; b, c, e and f 0 .6 sec . Tension calibration, a, b, d and e 10 gr ; c and f 40 gr . DISCUSSION Cross-innervation studies of fast and slow muscles that motor nerves cific type of a by

new,

regulate, among other characteristics,

myosin synthetized by the muscle .

qualitatively different

expression of the muscle support

(10) .

from experiments with

These drugs were found

This hypothesis

interpreted

influence on gene received

inhibitors of protein

to prevent

the spe-

The appearance of

species of protein was

postulating that motor nerves exert a direct

have shown

further

synthesis .

the development of denervation

Vol. 13, No . 12

changes

in mammalian muscle cells .

Fambrough med "In al

Actinomycin D ani FYbrillation Activity

(2) .

in denervated mouse

of extrajunctional

cholinergic

ance of Tetrodotoxin-resistant action the

resting membrane potential

mal

innervation .

changes ware protein the

and

it

reported

in

and

the effect

of

activity tation

results

be

nervated

animals

poration

into muscle

with this

antibiotic

finding

(9) .

D abolishes

induction of newly

of

This

interpre-

found between the

in early

injected de-

inhibition of urtdine

Grampp et

al

to mice

fibrillation

potentials

to other drugs

treated and

on

In our experiments, satisfactory .

only mild

diarrhea

Most of the animals

general

be explained

potentials because

some animals but

survived

to

intact .

condition of the treated animals

The highest dose employed in

is

l(ke tetrodotoxin which affects

the generation of action

the

intra-

This

treated denervated muscles maintained their excitability was

incor-

leaves the subthreshold activity un-

The blockage of fibrtllatton cannot

simple effect

can

in the generation of fibrillary

recorded subthreshold activity altogether .

in marked contrast

by a

This

(2) .

fibrillation potentials but Impaired

genetic

fibrtllatton

reported by

led us

development of

presented here show that

the similarity

and the period

changes

potential)

This

the

the section of a motor nerve .

delay in the appearance of

Actinomycin

D on

fibrillation .

involved

primarily based on

cellularly

actinomycin

indicating that

caused by

is

inhibitory

"pathway" possibly exists

investigate

Interpreted as

RNA and

another group of post-denervation

The

in

influence of the motor nerve

influence of the nerve .

formed proteins might

of

rise and overshoot of the action

reversibly affects

fall

the observed denervation

However since no

that more than one

activity .

the

of new species

regulatory

actinomycin D

the appear-

deprived of their nor-

the

fibrillary be

to the synthesis

reflected a regulatory

be thought

to mediate to

It was concluded that

related

maximum rate of

may

potentials

Grampp et

inhibited the

receptors,

in muscles

genome of the muscle cell .

effect was (e .g .

later confir-

muscle by

skeletal

According to these authors, actinomycin D

formation

on

finding described by

diaphragm (1), was

in organ cultured rat

vlvo"

This

1789

(0 .7 mg/kg)

tt was well

good condition

that during the complete experimental

tolerated .

despite

procedure the

produced

the fact

rats had been

1770

Actinomycin D and Fibrillation Activity

submitted

to five periods of ether anesthesia .

The fact brillation and

that

actinomycin D affected already established fi-

is of special

Interest

resting membrane potential

tions are not reversed could speculate that the development receptors .

(2) .

To

interpret

of fibrillation

Similar

since acetylcholine sensitivity

alterations

the turnover of

the turnover of those Miledi

vious

This

findings

showed that

interpretation

reported by

appearance of the earliest

is more

Grampp and

studied

In

D on

denervated frog

(12) .

These

no longer detected at

signs of

authors the time of

relnnervation whereas

the

two weeks

Ackn owledgements

We wish and

rapid than

by Bevan,

Increased sensitivity to acetylcholine persisted longer .

in

involved

is also consistent with pre-

Hfnes et al

fibrillation was

same condi

results, we

the effects of actinomycin

miniature end-plate potentials were (11) .

these

the formation of extrasynaptic

conclusions were drawn in which

under the

the molecules

potentials

underlying

in experiments

muscles

Vol . 13, No. 12

to thank

comments

Dr .

E.

De

Robertis for his

during the preparation of

helpful

suggestions

the manuscript .

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Fambrough,

Science ,

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2.

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Muchnik,

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