Histochemie 29, 1-7 (1972) �9 by Springer-Verlag 1972

Actomyosin ATPase Activity of Middle Ear Muscles in the Cat

ERIK TEIG and HANS A. DAHL

The Institute of Neurophysiology, the Ear, Nose and Throat Department, Rikshospitalet and the Department of Anatomy, Dental Faculty, University of Oslo, Oslo, Norway

Received November 12, 1971

Summary. When incubated for histochemical demonstration of actomyosin ATPase both the tensor tympani and the stapedius were found to contain two types of muscle fibres, one with high actomyosin ATPase activity, indicating a high speed of contraction, and one with low actomyosin ATPase activity, indicating a low speed of contraction. In the tensor tympani 59% and 41% of the muscle fibres had a high and low ATPase activity, respectively. The corresponding numbers in the stapedius muscle were 82 % and 18 %. These findings are discussed with reference to previous physiological and morphological studies.

Introduction

No conclusive evidence has so far been ob ta ined concerning the different muscle fibre types in the tensor t y m p a n i and s tapedius . Eru lkar , Shelanski , Whi t se l and Ogle {1964) found morphologica l and e leet rophysiological evidence for bo th twi tch and non- twi tch fibres in the tensor t y m p a n i of the cat. Besides typ ica l twi tch muscle fibres, F e r n a n d and Hess (1969) in bo th the tensor t y m p a n i and the s tapedius found muscle fibres which in thei r inne rva t ion and in te rna l s t ruc ture conformed to slow non- twi tch muscle f ibres in lower ver tebra tes . I n a physiologi- cal inves t iga t ion , Teig (1971 ) demons t r a t ed both fas t twi tch and slow twi tch mo to r uni t s in the tensor t y m p a n i of the cat, while only one type , wi th fast twi tch character is t ics , was found in the s tapedius . The present inves t iga t ion was under- t aken to see whether a h is tochemical s t u d y of ae tomyos in ATPase could assist in the ident i f ica t ion of the di f ferent muscle f ibre t ypes in the middle ear muscles and in the es t ima t ion of the propor t ion of the di f ferent f ibre types , since there is a close corre la t ion between the Ca++-act ivated myosin ATPase a c t i v i t y and cont rac t ion speed in di f ferent muscles (Bs163 I967; Gu th and Samaha , 1969; Buller, Mommaer t s and Seraydar ian , 1969).

Material and Methods

8 adult cats (2.3-4.1 kg body weight) were anaesthetized with sodium pentobarbital (30 mg/kg body weight) injected intraperitoneally, tracheotomized and immobilized in a special headholder which left the ears uninjured. A retroauricular incision through the skin and the platysma with subsequent removal of the tympanic membrane and the lateral part of the mastoid bulla gave wide exposure of the middle ear cavity. The tensor tympani and stapedius muscles were taken out, straightened with pins on a piece of cork, dipped in talcum powder (Moline and Glenner, 1964) and quenched in liquid nitrogen. The blocks were transferred to brass chucks and stored at --95 ~ C for periods up to some weeks before further processing (Pearse, 1968).

1 nistochemie, Bd. 29

2 E. Teig and H. A. Dahl:

Sections 10 ~m thick were cut in a Dittes Duspiva cryostat at --25 ~ C. Sections from the tensor tympani and stapedius were put together on the same slide to provide identical conditions during the incubation. Often, sections from the medial head of the gastrocnemius muscle were included for comparison. Incubation for actomyosin ATPase (pH 9.4) was performed according to Padykula and Herman (1955) as modified by Guth and Samaha (1969, 1970). Incubation time was 20 minutes at 37 ~ C. Prior to the incubation the sections were exposed to different solutions previously used to identify different types of muscle fibres (Guth and Samaha, 1969, 1970). The following preincubation procedures were used:

1. 1-5 minutes in 2% buffered formaldehyde, pH 7.6, at 4 ~ C (Prefixation). 2. Prefixation followed by alkaline 2-amino-2-methyl-l-propanol buffer (pH 10.4) for

15 minutes at room temperature. 3.0.1 M potassium acetate buffer, pH 4.1, for 4 minutes at room temperature. Some-

times, neighbouring sections were subjected to buffers of different pH (pH 1.9-4.5). 4. Prefixation followed by 0.1 M potassium acetate buffer pH 4.1 for 4 minutes at room

temperature. Control incubations were performed without substrate or with glucose-6-phosphate or

AMP replacing ATP as substrate in equimolar concentration. The sections were postfixed in 10% formalin containing 1% CaC12, dehydrated and mounted in Eukitt. The diameter of the muscle fibres was measured with an ocular micrometer (Zeiss). Counting of the different fibre types was performed on photographic prints.

Results

The Tensor Tympani Muscle. The muscle fibres of the tensor t y m p a n i could be d iv ided in two groups, one with high ATPase ac t i v i t y (59%; 521 out of 884 fibres counted) and one wi th low ATPase a c t i v i t y (41% ; 363 out of 884 fibres counted). Bo th types of muscle f ibres were evenly d i s t r ibu ted th roughou t the muscle (Fig. 1a-c) . I n the t u b a r region the muscle fibres were smal ler (Fig. lb ) t h a n in the more media l pa r t s of the muscle (Fig. 1 c). The fibres wi th a s t rong ATPase reac t ion had a much wider size range (5-30 ~m) t h a n those exhib i t ing a weak reac t ion (5-17 ~m) (Fig. 2A), bu t each group seemed to form one size popula t ion only. W h e n exposed to buffer solut ions in the acid range, the ae tomyos in ATPase reac t ion of the s t rongly reac t ing fibres decreased, mos t p ronounced in the sections exposed to the most acid buffer. Below p H 4.0 no ac t iv i ty persis ted, and no increase of ac t i v i t y in the weak ly reac t ing fibres ( "ac id r eve r sa l " of Gu th and Samaha , 1969) took place. Sections from the media l head of the gas t rocnemius incuba ted on the same slide showed increased ATPase a c t i v i t y of or ig inal ly low ATPase fibres a t p H 4.35 as repor ted b y previous inves t iga tors (Guth and Samaha , 1969). The ATPase ac t i v i t y of the s t rongly reac t ing fibres of the tensor t y m p a n i showed l i t t le resis tance to prein- cuba t ion in 2 % formaldehyde a t 4 ~ C and in alkal ine buffer p H 10.4.

The Stapedius Muscle. I n the s tapedius muscle the m a j o r i t y of the fibres gave a s t rong reac t ion for ac tomyos in ATPase (82%; 628 out of 769 fibres counted) , bu t sca t t e red small fibres showed a weaker reac t ion (18%; 141 out of 769 fibres counted). (Fig. 3b, cp. wi th Fig. l b and lc ) . The d iamete r of the ATPase s t rong fibres was 5-40 ~m and t h a t of the ATPase weak fibres 5-22 ~m (Fig. 2B). Nei ther in the ATPase s t rong nor in the ATPase weak group could any subgroups be ident i f ied on the basis of fibre size.

W h e n sections from the s tapcdius muscle were subjec ted to buffers in the acid range, the ac t i v i t y of the s t rongly reac t ing fibres decreased with decreasing p H jus t as in the tensor t ympan i , bu t a t p H 4 . 1 4 . 2 the ac t i v i t y in the weak ly reac t ing fibres increased (Fig. 3a and c). W h e n the p H of the p re incuba t ion

Actomyosin ATPase in Middle Ear Muscles 3

Fig. l a - c . Cat tensor tympani muscle. 10 ~zm cross sections. Actomyosin ATPase pH 9.4. a The greater par t of the muscle belly is shown. ATPase weak and ATPase strong fibres are evenly distr ibuted throughout the muscle. The areas indicated by frames are shown in greater magnification in (b) and (c). • 48. b Higher magnification from the tubar region of the muscle (lower left frame in a). The muscle fibers are generally small, and ATPase strong and ATPase weak fibres are evenly intermingled. • 290. c Higher magnification from the medial par t of the muscle (upper r ight frame in a). Bundles of fibres with a mixture of ATPase strong and weak fibres, are separated by connective tissue. The ATPase strong fibres are larger than the ATPase weak fibres, and larger than the ATPase strong fibres

in the tubar region. X 290

1"

4 E. Teig and H. A. Dahl: Actomyosin ATPase in Middle Ear Muscles

. o

o

0u

z

A Mtensor tympani B Mstopedius

200 ~ ATPose strong 40 ~ ATPoso strong 150~ 30 ~gop ~ 2g ~ ~

250 ATPose weok 50 AIPose weok

150~ 30 loop- 2o

50~ I0

J 2J5 gl 0 o g m ~5 20 o g ~g ~5 20

F ib re d i o m e l e r (pro] 2'5 3o 3'5 ~o

Fig. 2A and B. Histogram showing the diameter of ATPase strong and ATPase weak muscle fibres in the tensor tympani (A) and the stapedius (B) muscles of the cat. In (A) 993 fibres were measured in randomly selected areas from the medial and tubar part of one muscle.

In (B) 218 stapedius fibres from one muscle were measured

buffer was below 4.0 no reaction occurred, but the ATPase activity of the strongly reacting fibres of the stapedius was more resistant to prcincubation in formaldehyde or alkaline buffer than the ATPase activity of the strongly reacting fibres in the tensor tympani. Incubations without ATP as substrate or with AMP or glucose-6-phosphate replacing ATPase as substrate, gave no reaction in any of of the muscles.

Discussion

On the basis of the findings of Erulkar et al. (1964) Fcrnand and Hess (1969) and Teig (1971) one would expect the tensor tympani to contain three types of muscle fibres: one fast twitch, one slow twitch, and one s]ow non-twitch type. The present finding of only two types may be explained in two ways: the morphologically "slow non-twitch fibres" are actually of the twitch type, or they have a reaction for actomyosin ATPase identical to one of the twitch types.

Since high actomyosin ATPase activity has been correlated with high con- traction speed (Bs163 1967), the high ATPase activity fibres of the tensor tympani most probably represent the fast twitch motor units shown to have contraction times between 23 and 40 msec (Teig, 1971). In the gastrocnemius muscle, fast twitch motor units have contraction times between 16 and 30 msec (Burke, 1967), and show a high ATPase activity (Yellin and Guth, 1970). The fast twitch motor units of the tensor tympani consequently seem to be similar to fast twitch motor units of ordinary skeletal muscles with regard to both contraction velocity and actomyosin ATPase activity.

The ATPase-weak fibres of the tensor tympani probably constitute the slow twitch motor units with contraction times between 58 and 92 msec reported by Teig (1971). In contrast to low activity fibres in cat hind-limb muscles (Yellin and Guth, 1970) and to low activity fibres in the stapedius, the ATPase-weak fibres of the tensor tympani failed to be activated by preincubation in acid

Fig. 3a-c . Cat stapedius muscle. 10 [zm cross sections, a Incubated for actemyosin ATPase pH 9.4 after p re t rea tment with acid buffer (pH 4.1) show increased act ivi ty in originally low activity fibres. After acid preincubation capillaries react quite strongly and are seen both in the muscle (left par t of the figure) and in the facial nerve, which is seen to the right. The acid-activated fibre are scattered throughout the muscle. The area indicated by the frame is shown in greater magnification in Fig. 3c. x 48. b Plain actomyosin ATPase. A few low ATPase fibres are scattered between the high act ivi ty fibres. The diameter of the high ATPase fibres is greater than t ha t of the low ATPase fibres, and in general they are larger than the ATPase fibres of the tensor tympani, x 290. c Higher magnification of the area indicated by the frame in (a). b and c show the same area, and it is seen t ha t the low

activi ty fibres in (b) show high activity in (c) and vice versa, x 290

6 E. Teig and H. A. Dahl:

buffer. So far, however, the significance of the acid activated ATPase which is located primarily in between the myofibrils is unknown. In contrast to the plain or alkali-activated ATPase, which is believed to be relatively specific for aeto- myosin ATPase, it does probably not reflect the activity of any single enzyme (Guth and Yellin, 1971). Furthermore, since the contraction time of the slow twitch motor units of the tensor tympani is about the same as tha t of the slow twitch motor units of the gastrocnemius (40-87 msec) and of the soleus (55 to 110 msee) (Burke, 1967), the difference in reaction after subjection to acid buffer does not seem to reflect any major physiological difference.

So far, non-twitch muscle fibres have been shown to have an actomyosin ATPase activity different from tha t of the twitch fibres in the same muscles. In the frog, slow non-twitch fibres have very low actomyosin ATPase activity (Engel and Irwin, 1967), while in the atlantic hagfish they have a very strong activity (DaM and Nieolaysen, 1971). Since there is a good correlation between the contraction times of the two types of twitch motor units and the aetomyosin ATPase activity of the two types of muscle fibres in the tensor tympani, it is therefore possible that the multi-innervated, morphologically slow muscle fibres reported by Erulkar etal. (1964) and Fernand and Hess (1969) are identical with the slow twitch motor units reported by Teig (1971). In that case the fibre structure of the tensor tympani resembles that of the external eye muscles, where Baeh-y-Rita and I to (1966) in addition to fast twitch muscle fibres found slow twitch muscle fibres, which they thought to be identical with the multi- innervated muscle fibres described by Hess and Pilar (1963). These "slow multi- innervated twitch fibres" of the external eye museles had small junction poten- tials and a low resting potential comparable to tha t found in the supposedly slow muscle fibres of the tensor tympani by Erulkar et al. (1964). However, the evidence on this point is conflicting and the question of the contraction properties of muscle fibres with multiple nerve endings in the extraoeular muscles is not yet settled (Peachey, 1968; Hess, 1970).

In view of the considerations above one may conclude that the tensor tympani consists of : 1. fast twitch muscle fibres (contraction times between 23 and 40 msee) with high aetomyosin ATPase activity. These fibres probably have individual end plates; 2. slow twitch muscle fibres (contraction times between 58 and 92 msee) with low ATPase activity. The latter may have multiple nerve terminals.

One important reservation must be made, however, that possible non-twitch muscle fibres which could be multi-innervated, might have an actomyosin ATPase activity identical to tha t of one of the twitch types.

The interpretation of the findings in the stapedius muscle is more compli- cated since the possible existence of a small number of slow twitch motor units may have escaped notice during the physiological s tudy due to limitations in the recording technique (Teig, 1971). The majority of the fibres had a high ATPase activity and probably constitutes the fast motor units with contraction times between 14 and 39 msee encountered in the physiological s tudy (Teig, 1971).

A small proportion of the stapedius fibres had a low aetomyosin ATPase activity indicating a low contraction speed. These fibres could be identical with the fibres with multiple nerve terminals reported by Fernand and Hess (1969) and claimed by them to be slow. As emphasized by these authors more evidence is needed to determine whether these fibres are of the twitch or of the non-

Actomyosin ATPase in Middle Ear Muscles 7

twitch type. When subjected to p re t r ea tmen t with acid buffer, the low ATPase fibres of the stapedius were act ivated like low ATPasc fibres of slow twitch

motor uni ts of cat h ind l imb muscles (Guth and Samaha, 1969; Yellin and Guth, 1970), bu t as ment ioned above this reaction may not be related to the contract ion properties of a muscle fibre. Wi th the same reservation as made for the tensor t y m p a n i it is concluded tha t the stapedius muscle contains : 1. fast twitch muscle fibres {contraction times between 14 and 39 msce) with high actomyosin ATPase act ivi ty. These probably have individual end plates. 2. Fibres with low aetomyosin ATPase ac t iv i ty suggesting low contract ion speed bu t with otherwise u n k n o w n contract ion properties. At least some of them ma y have mult iple nerve terminals.

References Bach-y-Rita, P., Ito, F. : In vivo studies on fast and slow muscle fibers in cat extraocular

muscles. J. gen. Physiol. 49, 1177-1198 (1966). BArAny, M. : ATPase activity of myosin correlated with speed of muscle shortening. J. gen.

Physiol. 50, Suppl. 197-218 (1967). Bullet, A. J., Mommaerts, W. F. H. M., Seraydarian, K.: Enzymic properties of myosin in

fast and slow twitch muscles of the cat following cross-innervation. J. Physiol. (Lond.) 205, 581-597 (1969).

Burke, R. E.: Motor unit types of cat triceps surae muscle. J. Physiol. (Lond.) 193, 141-160 (1967).

Dahl, H. A., Nicolaysen, K. : Actomyosin ATPase activity in atlantic hagfish muscles. Histo- chemie (1971) in press.

Engel, W. K., Irwin, R. L. : A histochemical-physiological correlation of frog skeletal muscle fibers. Amer. J. Physiol. 213, 511-518 (1967).

Erulkar, S. D., Shelanski, M. L., Whitsel, B. L., Ogle, P.: Studies of muscle fibers of the tensor tympani of the cat. Anat. l~ec. 149, 279-289 (1964).

Fernand, V. S. V., Hess, A.: The occurrence, structure and innervation of slow and twitch muscle fibres in the tensor tympani and stapedius of the cat. J. Physiol. (Lond.) 2OO, 547-554 (1969).

Guth, L., Samaha, F. J.: Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. Exp. Neurol. 25, 138-152 (1969).

- - - - Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol. 28, 365-367 (1970).

- - Yellin, H.: The dynamic nature of the so-called "fiber types" of mammalian skeletal muscle. Exp. Neurol. gl, 277-300 (1971).

Hess, A.: Vertebrate slow muscle fibers. Physiol. Rev. 50, 40-62 (1970). - - Pilar, G.: Slow fibres in the extraocular muscles of the cat. J. Physiol. (Lond.) 169,

780 798 (1963). Moline, S. W., Glenner, G. G. : Ultrarapid tissue freezing in liquid nitrogen. J. Histochem.

Cytochem. 12, 777-783 (1964). Padykula, H. A., Herman, E. : Factors affecting the activity of adenosine triphosphatase and

other phosphatases as measured by histochemical techniques. J. Histochem. Cytochem. 8, 161-169 (1955).

Peachey, L. D.: Muscle. Ann. Rev. Physiol. 39, 401-429 (1968). Pearse, A. G. E.: Histochemistry, theoretical and applied, third ed., vo]. I, 759 pp. London:

J. & A. Churchill Ltd. 1968. Tcig, E. : Tension and contraction time of motor units of the middle ear muscles in the cat.

Acta physiol, stand. In press (1971). Yellin, H., Guth, L. : The histochemical

424-432 (1970). classification of muscle fibers. Exp. Neurol. 26,

Dr. Erik Teig Ear, Nose and Throat Department Rikshospitalet Oslo, Norway