Gunn, 1972) is associated with the type F fibres, and low activity with the type S

Transcription

1 J. Phy8iol. (1979), 295, pp With 3 text-figurea Printed in Great Britain RABBIT DIAPHRAGM: TWO TYPES OF FIBRES DETERMINED BY CALCIUM STRONTIUM ACTIVATION AND PROTEIN CONTENT BY PHYLLIS E. HOAR AND W. GLENN L. KERRICK From the Department of Physiology and Biophysics, University of Washington SJ-40, Seattle, Washington 98195, U.S.A. (Received 11 August 1978) SUMMARY 1. Submaximum Ca2+- and Sr2+-activated tensions were measured in functionally skinned fibres from the rabbit diaphragm. 2. The fibres could be classified into two types on the basis of activation by divalent cations. Type S fibres required a slightly higher [Ca2+] than [Sr2+] for halfmaximal activation of tension. Type F fibres required 2 times higher [Ca2+] than type S and 8-5 times higher [Sr2+]. 3. Sodium dodecyl sulphate disc gels of type F fibres showed protein band patterns similar to those of fast-twitch muscle; those of type S fibres were identical to those of slow-twitch fibres. 4. Sodium dodecyl sulphate disk gels of mixed, non-classified diaphragm fibres and an actual count of randomly selected fibres classified by Ca2+/Sr2+ tension characteristics showed the diaphragm to be composed of 60 % fast-twitch fibres and 40 % slow-twitch fibres. 5. The protein stoichiometries of the two fibre types are consonant with the accepted stoichiometries for skeletal muscle. 6. The percentages of type F and type S fibres in the diaphragm suggest that high adenosine-triphosphatase activity shown by histochemical techniques (Davies & Gunn, 1972) is associated with the type F fibres, and low activity with the type S fibres. INTRODUCTION Histochemically and physiologically, rabbit diaphragm fibres fall into two groups. One group has high ATPase activity and is thought to be of the fast-twitch type, whereas the other has low ATPase activity and is thought to be of the slow-twitch type (Davies & Gunn, 1972). Metabolism is predominantly anaerobic in the former type and aerobic in the latter (Davies & Gunn, 1972). The diaphragm fibres and their motor units can be classified into two types on the basis of the type of action potential observed in the cat phrenic nerve (Nail, Sterling & Widdicombe, 1969). In skeletal muscle of the cat the type of motor unit has been shown to be related to contraction time and to be associated with either a slow-twitch or fast-twitch fibre type (Burke,. 1968). In the present study, using a functionally skinned single fibre preparation we found two fibre types that differ in their protein composition and activation by divalent cations /79/ $ The Physiological Society

2 346 P. E. HOAR AND W. G. L. KERRICK METHODS Fibre preparation. New Zealand White rabbits were sacrificed by cervical dislocation. The diaphragm was excised, several 1 mm3 pieces of muscle from the costal region were removed, and functionally skinned single fibres were prepared for tension measurement according to the method of Kerrick & Krasner (1975). Since these fibres had no functional sarcolemma, the ionic environment surrounding the contractile proteins could be controlled while tension was monitored. 1 min Fig. 1. Protocol for measurement of percentage of maximum tension. Relaxed fibres were contracted to steady-state tension in a submaximal solution (pca 5.2), then in a solution generating maximal activation (pca 3-8), and then relaxed (pca 8). The ordinate is relative force and the abscissa time. Solution used for tenaion measurements. All solutions contained 70 mu-[k+ + Na+], 15 mmcreatine phosphate, 15 u. creatine phosphokinase/ml. solution, mm-imidazole, 7 mm- EGTA total, varying amounts of Cal+ or Sr2+ (pca = 8 to 3 and psr = oo to 3), 1 mm-mg2+, 2 mm-mgatp1-, and propionate as the major anion. We varied the amount of imidazole propionate to maintain computed ionic strength constant at 0-15 and ph buffered at 7 ( ± 0-02). The volumes of stock solutions of the above constituents required were determined by computer with ionic equilibria binding constants derived from the literature (Donaldson & Kerrick, 1975). Protocol. The skinned fibres were placed in a relaxing solution and viewed under a Wild binocular microscope with back lighting. Randomly selected single fibres were mounted in the forceps of a photodiode tension transducer apparatus similar to that of Hellam & Podolsky (1969), and tension was recorded while they were immersed sequentially in solutions containing different concentrations of either Cal+ or Sr'+, as follows. Each fibre was immersed first in a solution (pca > 8), then in a submaximal-contracting solution until steady-state tension developed, then in a maximal-contracting solution until maximum tension developed, and finally in a relaxing solution until steady-state relaxation was attained (Fig. 1). This 'stepping' protocol was used rather than a bracketing one, since both protocols gave the same results and more data could be collected by the faster 'stepping' protocol (Best, Donaldson & Kerrick, 1977). Four or five different pca data points (a series of contractions similar to the one in Fig. 1) were collected on one fibre followed by four or five psr data points for half of the fibres of each type attached to the tension transducers at one time. (Eight tension transducers were used.) The psr data points were collected before the pca points on the other half of the mounted fibres. The steady-state submaximal tension was expressed as a percentage of maximal activated tension (Fig. 2, Table 1). From the Cat+/Sr2+ activation data each fibre was classified, then removed from the apparatus and dissolved in boiling sodium dodecyl sulphate sample buffer (see below). Solutions containing dissolved fibres of the same type were pooled and run on sodium dodecyl sulphate polyacrylamide gels. Data obtained from Ca"+/Sr'+ activation and polyacrylamide gel electrophoresis were then correlated.

3 TWO TYPES OF RABBIT DIAPHRAGM SKINNED FIBRES TABLE 1. Means of percentages of maximum tensions + S.E. of the mean and sample size ( ) according to pca or psr and type of diaphragm fibre pca * Slow type P7 (4) (4) (12) (6) (12) (6) (6) 7-1+2*4 (5) *3 (4) 0 (1) C 0 C ~~~~ E X~~~~~~ Fast type psr ± 2-3 (12) '5 (7) ±+ 13 (13) (7) *5 + 3*7 (7) (6) 5-6 o (1) ±0'7 (2) 6-0 o (1) VA 100 Slow type (2) *8 (9) *2 (12) *3 (6) 40'1 + 5*4 (12) (12) (6) 0 (6) Fast type (5) (9) 50'3+330 (7) *9 (6) (7) (6) log [Ca2' or Sr2"] Fig. 2. Relationship between percentage of maximum tension and - log [Ca2+] or [Sr'+] for rabbit diaphragm type F (fast-twitch) and type S (slow-twitch) fibres. The standard error of the mean and sample size for each data point are given in Table 1. The ion concentrations at midpoint followed by (Hill n) for the curves drawn are: pca 5-36 (n = 1.52) and psr 5.53 (n = 2.20) for slow type fibres and pca 5-05 (n = 1.57) and psr 4 60 (n 2.01) for fast = type fibres. Fast type muscles: V, Ca; Y, Sr. Slow type muscles: 0, Ca; *, Sr. 347 Gel ekctrophoremi&. The fibres removed from the tension transducer were boiled in a heated sample buffer containing 0-01 nm-tris phosphate, ph = 6-8, 1 % sodium dodecyl sulphate, 1 % 86-mercaptoethanol, and 20% glycerol. The disk gels contained 10% acrylamnide (Bio-Rad), 0-1% N,N'-bismethylene acrylamide (Bio-Rad), 0-1 % sodium dodecyl sulphate, 6% glycerol, 22 mg ammonium persulphate per 55 ml. total volume, 73 Iu1. TEMED (Bio-Rad) per 55 ml. total volume, and 0.1 mm-tris phosphate, ph = 6*8. After electrophoresis the gels were stained

4 348 P. E. HOAR AND W. a. L. KERRICK in a solution containing 0-1 % Coomassie Blue G 250, 45% methanol, and 9-3 % acetic acid, and then destained by diffusion in a solution containing 5% methanol, 7-5% acetic acid. Molecular weights were determined from known standards: phosphorylase (100,000 daltons), bovine serum albumin (68,000 daltons), actin (45,000 daltons), carbonic anhydrase (29,000 daltons), troponin-c (18,000 daltons), and dogfish parvalbumin (10,500 daltons). Fibre proteins were identified by comparison with proteins previously purified from rabbit fast- and slow-twitch skeletal muscle. TABLE 2. Relative moles of proteins determined from sodium dodecyl sulphate polyacrylamide gels* Mixed Fast Slow Mol. wt. fibres$ fibreal fibres LC3Ot 14, LC2+troponin-C (f+s) 18, Troponin-I (f+s) 23, LC1f 25, LCI. 27, Tropomyosin + troponin-t (f+ s) 36, , , * Data is based on the assumption of equal dye binding per g protein. Actin is not included in this Table due to comigration with creatine phosphokinase from the physiological solutions used to prepare the skinned fibres. t LC, Myosin light chain; f, fast; s, slow. T Relative to 1 mole of LC1I. Relative to 2 mole of LC1,. Stoichiometric determinations. The polyacrylamide gels were scanned in a densitometer at 570 nm and the peak areas determined with a Numonics Model 1224 electronic graphics calculator. For a particular sample of mixed diaphragm fibres, four gels of increasing sample load were run and the peak area of each protein was plotted against the particular sample load. These plots gave straight lines through the origin with R' values > 0-99 for each protein listed in Table 2. Ratios of the slopes of these lines divided by the appropriate molecular weight give relative molar values of the muscle proteins. It is not necessary to determine what a particular area corresponds to in terms of 4ag protein when equal dye binding by weight of protein is assumed. For each of two separate samples of mixed diaphragm, we determined the ratios of proteins relative to LC1I (light chain), and then averaged the two ratios to obtain the values for mixed diaphragm fibres (listed in Table 2). We used the ratios of the area of each protein band divided by the appropriate molecular weight to obtain the relative moles for the proteins of the fast and slow fibres since one gel was used for each fibre type. Curve fitting. The curves drawn in Fig. 2 to fit the data were determined by the Hill equation (Hill, 1913). /%T [Ca] n 100 [Ca]" + 10-R' where % T is the percentage of maximum Ca2+_ or Sr2+-activated tension and n and R are constants. We used a computer program to find values of the parameters n and R that minimized the sum of the squared deviation between % T and the calculated values of % T. The pca required for 50% tension is given by R/n. RESULTS Ca2+/Sr2+ activation. Two types of diaphragm fibres distinct in their activation by Ca2+ and Sr2+ were found (Fig. 2). The standarderror of the mean and sample size for each data point shown in Fig. 2 are given in Table 1. Type F fibres were more sensitive to Ca2+ than to Sr2+, requiring approximately 3 times higher Sr2+ concen-

5 TWO TYPES OF RABBIT DIAPHRAGM SKINNED FIBRES 349 tration than Ca2+ for half-maximal activation. In contrast, type S fibres were activated by slightly lower concentrations of Sr2+ than Ca2+. A comparison of the [Ca2+] and [Sr2+] required for half-maximal activation shows that the type F fibres required 2 times higher [Ca2+] than type S and 8-5 times higher [Sr2+]. A Actin TM Mixed fibres Fast fibres Slow fibres LC2f LC2, TN I LC'f LCjs B Actin Adductor (fast) :'., Soleus (slow) - ---S-*Jti\@Z-* Fig. 3. A, densitometric scans of sodium dodecyl suphate poly~acrylamide gels of mixed rabbit diaphragm fibres from homogeneous muscle, type F (fast) diaphragm fibres, and type S (slow) diaphragm fibres. B, scans from rabbit adductor magnus (fast-twitch) and soleus (slow-twitch) fibres. Note: Troponin-C comigrates with myosin light chain 2. Gels were scanned to about 100,000 molecular weight. LC, myosin light chain; f, fast; s, slow; TNI, troponin-i; TM, tropomyosin; TNT, troponin-t. Fibre proteins. The densitometric scans of sodium dodecyl sulphate polyacrylamide gels in Fig. 3A show clearly that type F (fast) diaphragm fibres can be distinguished from type S (slow) by their protein band patterns. Furthermore, each protein band in the mixed diaphragm sample can be accounted for in one of the two fibre types. Comparison of the protein band patterns in Figs. 3A and 3B shows a striking resemblance between the diaphragm type F and adductor magnus (fast-twitch) fibres and between the diaphragm type S and soleus (slow-twitch) fibres.

6 350 P. E. HOAR AND W. G. L. KERRICK In Table 2 the stoichiometry of fibre proteins is given relative to 1 mole myosin LC1f for the fast type or mixed sample of fibres and relative to 2 mole myosin LC15 for the slow type. Values for actin are not given since the tension solutions used contained creatine phospokinase, which comigrates with actin on these gels. TABiL 3. Distribution of the fibre types % total fibres mounted % determined on (number) SDS gels* TypeFfibres 60 (27) 56 Type S fibres 40 (18) 44 * See text for calculation. SDS, sodium dodecyl sulphonate. Distribution of fibre types. The relative distributions of the two fibre types in the rabbit diaphragm were estimated by two methods and compared (Table 3). In one method, a count was taken of the fibres of each type encountered during the classification of the fibres based on their Ca2+ and Sr2+ activation of tension. In the other method the relative distributions of the two fibre types were determined from the relative concentrations of fast and slow myosin according to the following equations: % fast type = LCX,+LC3. x100, LC1f + LC3f + LC18 % slow type = Mu LC~LC8X 100, where LC1f, L03f, and LCj. are the values given for mixed fibre types in Table 2. Fast myosin enzymes are known to be predominately homodimers containing either two LCjf or two LC3f (Holt & Lowey, 1977) and slow myosin to contain two LCJ" (Weeds, 1976). Thus the relative amount of fast and slow myosin can be determined by the above equations. As one can see in Table 3, both methods gave nearly the same relative distribution of fibre types. DISCUSSION The type S and type F fibres of the diaphragm are qualitatively similar in activation by divalent cations to the relatively homogeneous slow-twitch (soleus) and fast-twitch (adductor magnus) rabbit skeletal muscles, respectively (Kerrick, Secrist, Coby & Lucas, 1976). Ebashi, Endo & Ohtsuki (1969) showed that crude actomyosin preparations of fast-twitch skeletal muscle were 34 times more sensitive to [Ca2+] than [Sr2+], whereas slow-twitch muscles were approximately 7 times more sensitive to [Ca2+] than [Sr2+]. Our tension measurements are in agreement only qualitatively with this observation; a slightly greater sensitivity to [Sr2+] than to [Ca2+] was seen for type S fibres (identified as slow-twitch), and a greater relative sensitivity to Ca2+ than Sr2+ for type F fibres (identified as fast-twitch). The differences in sensitivity to [Ca2+] and [Sr2+] between skinned fibres and actomyosin ATPase may be related in part to the differences in solutions used, the measurement of activation (superpercipitation vs. tension), or muscle types compared. However,

7 TWO TYPES OF RABBIT DIAPHRAGM SKINNED FIBRES 351 since the [Ca2+] and [Sr2+] activation properties of type F and type S fibres compare quite well with those of adductor magnus (fast-twitch) and soleus (slow-twitch) muscles, respectively (Kerrick et al. 1976), the difference in fibre type between diaphragm and other skeletal muscle would not seem to be a plausible explanation. It may be that the troponin-tropomyosin systems responsible for the [Ca2+] and [Sr2+] activations of the two fibre types differ in selectivity properties (Ebashi et al. 1969) or that some other mechanism is involved in Ca2+ activation. The stoichiometry measurements presented here (Table 2), which assume equal dye-binding properties of the proteins, are in general agreement with those reported by Potter for rabbit fast skeletal muscle (1974). It is difficult to make an exact comparison between our data and his due to the omission of myosin LC3f and LC11 in his data and the comigration of LC2 and troponin-c as well as tropomyosin and troponin-t bands and the omission of actin in our data. In the present study, comparison of stoichiometries should give a good relative estimate of any differences between the two fibre types; one would not expect the dye-binding properties of homologous proteins to be very different between fibre types. The data presented here show that the two fibre types in the diaphragm, like those of adductor and soleus muscle (Kerrick et al. 1977), contain proteins that differ in molecular weight but not in stoichiometry. This finding supports the hypothesis that striated muscle, regardless of type, contains approximately the same stoichiometric amounts of contractile and regulatory proteins. The relative distributions of the two fibre types in the rabbit diaphragm determined in the present study appear to correlate quite well with the percentages of fast-twitch and slow-twitch fibres calculated histochemically by Davies & Gunn (1972) on the basis of adenosine-triphosphatase activity. Therefore, one would conclude that the type F fibres (classified as fast-twitch) correspond to the fibres with high myosin ATPase activity reported in the rabbit diaphragm and the type S fibres (classified as slow-twitch) with those of low myosin ATPase activity. We thank Dr Dean Malencik in the Department of Biochemistry for making available to us protein samples from fast- and slow-twitch rabbit skeletal muscle and Roberta Coby and Barry Hill for technical assistance. This research was supported by grants from the Muscular Dystrophy Association, American Heart Association (76-842), and the U.S. Public Health Service (AM 17081). Dr Hoar was supported by a National Research Service Award (HL 05527) and a fellowship from the Muscular Dystrophy Association. REFERENCES BEST, P. M., DONALDSON, S. K. B. & KERRICE, W. G. L. (1977). Tension in mechanically disrupted mammalian cardiac cells: Effects of magnesium adenosine triphosphate. J. Phy8iol. 265, BURKE, R. E. (1968). Firing patterns of gastrocnemius motor units in the decerebrate cat. J. Phy8i1. 196, DAVIES, A. S. & GuxN, H. M. (1972). Histochemical fibre types in the mammalian diaphragm. J. Anat. 112, DONALDSON, S. K. B. & KERRICK, W. G. L. (1975). Characterization of the effects of Mg"+ on Ca'+- and Sr2+-activated tension generation of skinned skeletal muscle fibers. J. gen. Physiol, 66, EBASI, S., ENDO, M. & OHTSUKI, I. (1969). Control of muscle contraction. Q. Rev. Biophy8. 2,

8 352 P. E. HOAR AND W. G. L. KERRICK H]:LM, D. C. & PODOL&KY, R. J. (1969). Force measurements in skinned muscle fibres. J. Physiol. 200, HnL, A. V. (1913). The combinations of haemoglobin with oxygen and with carbon monoxide. I. Biochem.J. 7, HOLT, J. C. & LOwEY, S. (1975). An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical-chemical and immunological characterization of the light chains. Biochemistry, N.Y. 14, KERRICK, W. G. L., HoAR, P. E., MALENCIK, D. A., POCINWONG, S., COBY, R. L. & FISCHER, E. H. (1978). Calcium ion activation: Characterization in skinned skeletal and cardiac muscle fibers. Proceedings Third Joint US-USSR Symposium on Myocardial Metabolism Williamsburg, Virginia, May 9-11, 1977, National Institutes of Health, pp KERRICK, W. G. L. & KRASNER, B. (1975). Disruption of the sarcolemma of mammalian skeletal muscle fibers by homogenization. J. apple. Physiol. 39, KERRICK, W. G. L., SECRIST, D., COBY, R. & LucAs, S. (1976). Development of difference between red and white muscles in sensitivity to Cal+ in the rabbit from embryo to adult. Nature, Lond. 260, NAIT, B. S., STERING, G. M. & WIDDICOMBE, J. G. (1969). Some properties of single phrenic motoneurones. J. Physiol. 200, P. POTErR, J. D. (1974). The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils. Archs Biochem. Biophys. 162, WEEDS, A. G. (1976). Light chains from slow-twitch muscle myosin. Eur. J. Biochem. 66,