The electrophysiological expression of Ca2+ channels and of apamin sensitive Ca2+ activated K+ channels is abolished in skeletal muscle cells from mice with muscular dysgenesis

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1986

The electrophysiologicaI expression of Ca*+ channek and of apamin sensitive Ca*+ activated K+ charnels is aboIished in skeletal muscle cek from mice with muscular dysgenesis G. Romey, F. Rieger”, J.F. Renaud, M. Pincon-Raymond* and M. Lazdunski Centre de Biochimie Centre National de la Recherche Scientifique Part Valrose, 06034 Nice Cedex France *Groupe de Biologie et Pathologie Neuromusculaires INSERM U.1.53, 17 rue du Fer-a-Moulin 75005 Paris, France Received March 19, 1986 SUMMARY’. Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarisation (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action tentials which were never followed by a.h.p.‘s. Voltage-clamp experiments have shown p”i) that Na+ channel activity was identical in mutant and control muscles and (ii) that the activity of fast and slow Ca 2+ channels was nearly absent in the mutant. @ 1986 Academic

Press,

Inc.

INTRODUCTION.

Muscular

dysgenesis

(mdg) is a spontaneous recessive

in the mouse. The disease is characterized contractile

activity

in skeletal

contractions,

properties have shown internal

sarcoplasmic contraction

limb and diaphram

of myotubes,

reticulum

they exhibit

namely,

passive

or evoked are able to

or electrically

electrical

membrane

(4-10). Ultrastructural present

muscles

centrally-located

(SR), disorganization

clots. Moreover

abnormalities

nuclei,

of the 2 striations

dilatation

We have observed recently

normal mice. This observations

of the of the of

system

(11, 12, 13).

that there was a very large decrease of the number of

binding sites for PN 200-110, a dihydropyridine muscles

studies

and occurence

membranous couplings between SR and the T-tubule

known as triads are rare and disorganized

channels (12), in striated

normal

and induced action potentials

that mdg/mdg

structure

but they do not show any spontaneous

although

and spontaneous

by a total lack of spontaneous

muscles (1-3). In culture, mdg/mdg myoblasts

divide, fuse and form myotubes stimulated

lethal mutation

derivative

which is a marker

of Ca2+

of the mdg/mdg mutant as compared to muscles of strongly

suggested

that skeletal

muscles in mice with 0006-291X/86 $1.50

935

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Copyright 0 1986 rights of reproduction

by Academic Press, Inc. in any form reserved.

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muscular dysgenesis had lost most of their voltage-dependent work

presents

important skeletal

electrophysiological

data showing

link in excitation-contraction

coupling

that

Caz+ channels. The present Ca3+ channels,

probably

an

(14, 15), are missing in cultures

of

muscles of the mdg/mdg mutant. This nearly total absence of Ca2+ channels is

accompanied by other changes in electrophysiological MATERIALS

properties

of mdg/mdg muscles.

AND METHODS

Cd culture. Primary cultures of skeletal muscle cells from l&day-old to 19-dayold mouse embryos +/mdg? or mdg/mdg were prepared as previously described (8) using front and bindlimbs muscles. We refer to the phenotypically normal littermates of mdg/mdg newborns as +/mdg?, since +/+ and +/mdg mice are indistinguishable. Muscles were dissected free of their skin and paws, washed and trypsinized 30 min in a medium containing 116 mM NaCl, 5.3 mM KCl, 8 mM NaH2P04, 22.6 mM NaHC09, 1 mM glucose at pH 7.4 supplemented with 0.1 % trypsin (Gibco). Isolated cells were grown in Hz1 Dulbecco’s modified Eagle medium supplemented with 10 % horse serum, 1.25 % chick embryo extract, 200 units/ml of penicilin and 50 &ml of streptomycin. Cells were plated in gelatin-coated 35 mm diameter dishes (Falcon) at a density of 105 cells/cm2. Cells in culture were maintained at 37“C in a water-saturated atmosphere of air/C02 (95/5). Fused myoblasts into myotubes were used 3 to 8 days after plating. Myoballs were obtained by adding colchicine (10 nM) to the culture medium when the cells reached the early myotubular stage of development. Ekctrophysiology and amtraction. Culture dishes containing +/mdg and mdg/mdg myotubes or myoballs were directly used after the replacement of the culture medium by a specific external solution. For Na+ current measurements, the external solution contained (in mM) : 130 NaCl, 25 tetraethylammonium chloride (TEA), 1.8 CaC12, 0.4 MgC12, 5 glucose. This solution was buffered at pH 7.4 with 10 mM HEPES/tetramethylammonium hydroxide. For Ca3+ current measurements, the external solution contained (in mM) : 140 TEA, 2.5 CaC12 or BaC12, 0.4 MgCl2, 5 glucose. This solution was buffered at pH 7.4 with 10 mM HEPES/KOH. Intracellular recordings on myotubes were made using conventionnal glass microelectrodes filled with 3 M KCl, which had resistances ranging from 20 to 30 MR. The microelectrode was connected to an electrometer amplifier (WPI M 707) allowing simultaneous injection of current and recording through the same microelectrode. Rounded cells (myoballs) were voltageclamped by the whole-cell variant of the patch-clamp technique (16). The pipette solution contained (in mM) : 140 CsCl, 5EGTA and 10 HEPES buffered at pH 7.2 using CsOH. The osmolarity of the solutions was ajusted with sucrose to 300 mosM. Patch pipettes (2-6 Ma) were connected to the head stage of the recording apparatus (RK 300, Bio-Logic, Grenoble, France). The Na+ currents were low-pass filtered at 10 kHZ, sampled at 25 kHZ; the Ca2+ currents were low-pass filtered at 2 kHi! sampled at 5 kHZ. These signals were sampled, stored and analyzed on a Plessey 6220 system (Irvine, California, USA). Contractions were recorded simultaneously with the electrical activity by using the combination of a Sony TV camera and a simplified real time TV image analyzer based on an Apple II microcomputer (17). RESULTS AND DISCUSSION’. We have confirmed

that both +/mdg? myotubes taken as

controls

to produce

and mdg/mdg

electrical

stimulations

Electrical

activity

were

(6, 7). However,

and contraction

Action potentials after-hyperpolarization

myotubes

in +/mdg?

capable

action

potentials

only +/mdg? myotubes could contract

under

(Fig. 1A).

were uncoupled in mdg/mdg myotubes (Fig. IB). myotubes

were

always

followed

(a.h.p.1 (Fig. 1A). This a.h.p. resembled 936

by a long lasting

that recorded

in rat

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Fl. 1. Action potentials (upper traces) and contractions (lower traces) evoked bg anodal break stimulation in a 130 mM Na+ external solution containing 1.8 mM Ca +. A. In +/mdg? myotube, the action potential induced a contraction and was followed by a longlasting after-hyperpolarization (a.h.p.). B. In mdg/mdg myotube, the a.h.p. was absent and no contractile activity was associated with the action potential. C. Same +/mdg? myotube myotube

as in A, selective block of the a.h.p. by as in B; absence of effect of 10 nM apamin.

myotubes

in cultures

(18, 19). In rat myotube

activation

of a Ca2+-dependent

nium (TEA) and specifically a.h.p. following

K+ conductance

the action potential

in +/mdg? myotubes

of a contraction

Ca2+ concentration

during

the action potential

and insensitive

potential

Ca2+ concentration either

to be due to the to tetraethylammo-

apamin (19, 20). The

is also resistant

to TEA and

of an a.h.p. due to a Ca2+-activated

indicate that there is an increase of the

the electrical

activity

of +/mdg?

in mdg/mdg myotubes was never followed

Ca 2+-dependent

myotubes. by an a.h.p.

K+ channel or to the fact that the

does not increase in the mutant muscle during the action

because of the absence of Ca2+ release from the SR system Ca 2+ channels in the T-tubular

because of the absence of voltage-dependent the experiments

mdg/mdg

to apamin Fig. ID. The absence of a.h.p. in mdg/mdg myotubes could be

due to the lack of a functional internal

D. Same

which is insensitive

blocked by the bee venom neurotoxin

K+ channel and the existence

Conversely,

apamin.

the a.h.p. is known

blocked by 10 nM apamin (Fig. IC). The existence

cytosolic

10 nM

illustrated

in Fig. 2A,B, action potentials solution containing

or/and

system. In

were recorded in a 2.5 mM

Ca2+, Nat-free

external

140 mM TEA. Under these conditions, Na+

channel activity

was eliminated and TEA-sensitive K+ channels were blocked. Therefore,

the recorded action potentials were expected to be generated by the activation of Ca2+ channels. Under these conditions it was still possible to evoke slow Ca2+ action 937

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J

2. Action potentials (upper traces) and contractions (lower traces) evoked by anodal ZXstimulation in a 2.5 mM Ca 2+ (A and 8) or Ba2+ (C), Na+-free externaI solution containing 140 mM TEA. A. In +/mdg? myotube, the slow Ca2+ action potential was followed by an a.h.p. and induced a contraction. B. In mdg/mdg myotube, neither the anodal break stimulations or depolarizing pulses of current could trigger any Ca2+ action potential or contraction. C. In +/mdg? myotube, replacement of the external Ca2+ by Ba2+ led to a complete block of the contraction and a slowing down of the repolarizing phase of the action potential caused by inhibition of the a.h.p.

potentials

in +/mdg?

contractions

myotubes

and were

seemed

and there

followed

to record

any Ca*+ action

was no contraction

to be linked

replacement

to the

experiments.

were quite similar Two excitable two

in +/mdg?

voltage

ions

myotubes,

through

Ca2+ between

strong

it was impossible

and a.h.p.‘s

2B). In +/mdg?

particularly

The first ( s -60 mV).

20 msec) and rapidly

differ

from mdg/mdg the contractions

channels

since

electrical

the

activity

inactivating

have a higher

phase with a time-to-peak

voltage

(with

with a time constant threshold

and time

described

of

(26). These and by their

by a low threshold between

level of 10 and

of 20 to 30 msec. The second type ( > -30 mV), a slow activating

msec and a very slow decaying 938

in a variety

dependence

a time-to-peak

level of activation

of about 70-100

of the Na+ currents

muscle cells in culture

is characterized

activating

in voltage

(Fig. 3A,B).

in rat skeletal

type of Ca*+ current

were analyzed

and kinetics

have been recently

by their

It is rapidly

myotubes

dependence myoballs

types of Ca2+ currents

cells and (21-25)

of Ca2+ current

potentials

and mdg/mdg

and mdg/mdg

types of Ca2+ currents

activation

(Fig. of Ca2+

in +/mdg?

The amplitude,

distinct

pharmacology.

triggered

(Fig. 2C).

Na+ and Ca2+ currents clamp

entry

potentials

by a.h.p%. Conversely,

Ca+ by Ba 2+ led to the uncoupling

of external

and contraction

Ca*+ action

systematically

with the same conditions myotubes

(Fig. ZA). These

phase.

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A-B. Voltage clamp analysis of the Na+ currents in +/mdg? (A) and mdg/mdg (B) Is bathed in a 130 mM Na+ solution containing 1.8 mM Ca2+ and 25 mM TEA. Families of Na+ currents associated with step depolarizations to -55, -50, -45, -40, 35 mV from a holding potential VH = -90 mV. C-D-E. Voltage clamp analysis of the Ca2+ currents in +/mdg? (C,D) and mdg/md (E) myoballs bathed in a 2.5 mM Ca2+, Na+-free solution containing 140 mM TEA. (C,D f Same CaZ+ currents records with different time scales from +/mdg? myoballs. A fast activating current occured for a step depolarization to -35 mV from VH = -90 mV. A second very slow current appeared with a step to Ca2+ currents were

-15 mV.

E.

Under

the same conditions strongly depressed

Fig. 3C,D,

that

shows

One type of Ca2+ current current.

of stimulations, both in mdg/mdg myoballs.

the two types of Ca2+ currents is very similar

The other Ca*+ current

in rat muscle but with a slower

fast

and

slow

activating

coexist in +/mdg? myoballs.

to the above described

fast activating

is similar to the second slow type of current activation

These two types of Ca*+ currents

kinetics

are drastically

(time-to-peak decreased

Ca*+

described

of about 150 msec).

and hardly

detected

in

mdg/mdg myoballs (Fig. 3E). The electrophysiological currents

in mdgfmdg skeletal

previous biochemical

results

have shown a nearly complete

muscles in culture.

absence of Ca*+

This result agrees perfectly

with our

data (12, 13) showing a net (> 80 %) decrease of the specific binding

sites for PN 200-l 10, a specific

blocker of one of the two classes of Ca2+ channels (19)

in mdg/mdg skeletal

muscles as compared to +/mdg? muscle ; it also agrees with more

recent observations

made by others

(27). Taken together

with our previous

these results may suggest (i) that there is a direct relationship ce of triads

and of Ca*+ channels

in the mdg/mdg 939

work

(12)

between the disappearan-

mutant,

(ii) that the lack of

Vol. 136, No. 3, 1986

contraction

8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the paralysed mutant is directly linked to the absence of Ca2+ channels. It

will also be important channels disappears

to study in future investigations in skeletal

muscles

remains at the same level of concentration

why the I,4 dihydropyridine

of the muscular

dysgenesis

mutant

Ca2+

while

it

in the control and in the heart of the mutant

(12, 13).

ACKNOWLEDGEMENTS. This work was supported by the ‘Association les Myopathies’ and the ‘Centre National de la Recherche Scientifique’.

de Lutte contre

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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