102 ( 1982)
Fast to Slow Transformation of Fast Muscles in Response to Long-Term Phasic Stimulation F. A. SRETER, K. PINTER, F. JOLESZ, AND K. MABUCHI’ Department of Muscle Research, Boston Biomedical Research Institute; Department of Neurology, Massachusetts General Hospital; and Department of Neurology, Harvard Medical School, Boston, Massachusetts 02114 Received May 18, 1981 Indirect stimulation via the peroneus nerve, 60-Hz stimulus trains of 2.5 s duration, applied every 10 s for 5 weeks transformed rabbit fast-twitch extensor muscles to the slow type as judged by histochemistry, myosin ATPase activities, and electrophoresis on polyacrylamide gels. Electrophoresis of single fibers from the stimulated muscle under dissociating conditions showed the presence of both fastand slow-type light chains within the same fiber; under nondissociating conditions the slow-type myosin isoxyme predominated. Histological examination of the 5week stimulated muscle showed no signs of regeneration (de eovo fiber formation), suggesting reprogramming of existing fibers. These experiments show that fast to slow transformation is produced not only by continuous stimulation at a low frequency but also stimulation by trains at a higher frequency, indicating that muscle fiber transformation depends more on the total activity rather than the pattern of stimulation.
INTRODUCTION Reprogramming of the synthesis of the fiber type-specific contractile proteins, changing the activity of the sarcoplasmic reticulum, and shifting the ratio of glycolytic to oxidative enzymes can be achieved by stimulating the motor nerves of fast-twitch mammalian muscles at low frequencies ( 13- 17). Attempts to convert a slow muscle into a fast one by denervating Abbreviations: SDS-sodium dodecyl sulfate, PI+pyrophosphate. ’ The permanent address of Dr. Pinter is Department of Biochemistry, ELTE, Budapest, Hungary. The present address of Dr. Jolesz is Department of Physiology, Harvard Medical School, Boston, MA 02114. We thank Dr. J. Gergely for helpful discussions. This work was supported by grants from the National Institutes of Health (AG-2103, HL-23967, HL-5949) and the Muscular Dystrophy Association, Inc. 95 OOl4-4886/82/010095-08$02.00/O [email protected]
Q 1982 by Acadcmii F%ss, Inc. of rcpruduction in any form reurvcd.
and directly stimulating with brief high-frequency trains have led to confusing results (5, 7). We now report that long-term indirect stimulation of the fast extensor tibialis anterior muscle with 60-Hz trains of 2.5 s duration leads to transformation to the slow type as evidenced by changes in the myosin ATPase activity and the type-specific light chain and isozyme pattern, accompanied by a decrease in the activity of the sarcoplasmic reticulum and a shift toward an oxidative metabolic enzyme pattern. MATERIALS
A continuous stimulus pattern (60 Hz, 2.5 s duration, every 10 s) was imposed using implanted platinum electrodes which were sutured to the fascia near the peroneus nerve. The wires were led subcutaneously to the back of the animal and then to an external stimulator (Model S88, Grass Instr., Quincy, Mass.). Rabbits were placed in a cage supplied with a frontal transverse bar, to which the rabbit was attached by a collar several days before aseptic surgery. This arrangement permitted the animals to eat, drink, and sleep comfortably, but prevented them from reaching their back. During the stimulation period (to 5 weeks) the body weight slightly increased. After completion of the experiment the animal was killed, and the tibialis anterior muscles quickly dissected from both hind limbs for biochemical and histochemical studies. The weight of the stimulated muscle was approximately the same as that of the nonstimulated contralateral muscle, without any sign of atrophy. This was in contrast to our earlier, continuous, low frequency (10 Hz) stimulation experiments (17) in which the stimulated muscles weighed always 30 to 50% less than their contralateral muscles. Muscle fibers were characterized by methods described earlier, viz., by changes in the myosin ATPase activities, light-chain complement as shown by SDS-gel electrophoresis (6) isozyme pattern obtained by nondissociating (PPi) gel electrophoresis (4), and histochemical myosin ATPase and Ca’+-uptake assays (1, 9, 10). Single fibers were isolated and used for gel electrophoresis essentially as described by Mabuchi et al. (11). RESULTS Four rabbits were used in these experiments and in all cases we observed various degrees of fast to slow transformation. We report here the longest experiment, in which the transformation was most striking. Muscle sections stained for ATPase activity (Fig. 1) after acid preincubation showed a marked increase of fibers containing slow-type myosin. An increased oxidative activity was also apparent (diaphorase staining) as
FIG. 1. Hiitochemistry of the stimulated and unstimulated tibialii anterior muscles in rabbit. Each panel contains a section of a stimulated (upper two-thirds) and an unstimulated (lower one-third) tibia& anterior. A-CaMg-AT%se staining in the presence of IO?6 ethanol (10). Fibers having fast-type myosin appear dark. B-Ca-ATPase after acid preincubation at pH 4.3 (I), slow-type myosin stains dark. C-diaphorase (NADH-tetrazolium reductase) staining (12), indication of mitochondrid oxidative enzyme activity.
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FIG. 2. SDS-gel electrophoretogram of myosins isolated from various rabbit muscles. Electrophoresis on slab gel was carried out according to the method of Laemmli (6) using 14% separating gel. Gels were stained in 0.1% Coomassie brilliant blue (R 250) dissolved in 50% methanol and 10% acetic acid. Myosin loads are 10 (1, 2, 4, and 6) or 20 pg (3 and 5). Myosins of I-adductor magnus muscle (fast type); 2 and 3-tibialis anterior, unstimulated control; 4 and 5-tibialis anterior, stimulated; 6-soleus (slow type) muscle. Gels 3 and 5 were overloaded to demonstrate the slow-type light chains in unstimulated tibialis anterior (3) and their increased presence due to stimulation (5).
previously reported in other types of experiments (13, 17) leading to fast to slow transformation. The Ca2+ uptake on cryostat sections (9) in the contralateral and stimulated tibialis anterior muscle was 17.3 and 5.9 pmol Ca2sp/g protein/ min, respectively. These results were in good agreement with the histochemical findings (Fig. 1) showing significant increase in the type 1 fiber population in the stimulated muscle. The values for the Ca2+-activated myosin ATPase activity (measured in 0.025 M KCl, 10 mM CaCl,, 2.5 mM ATP, and 50 mM Tris, pH 7.6; reaction started by the addition of 10 to 15 pg/ml protein, 25°C) decreased from 0.6 in the contralateral tibialis anterior to 0.3 in the stimulated muscle (unit: pmol/mg/min). The transformation was clearly reflected in the light-chain complement of stimulated muscle myosin shown by SDS-gel electrophoresis (Fig. 2). Fast and slow type myosins show distinct patterns under these conditions.
FIG. 3. SDS-gel electrophoretogram of myosins and single fibers isolated from various muscles. The isolation and preparation of single fibers for electrophoresis was described earlier (9). I-myosin from adductor magnus; 2-single fiber from adductor magnus; 3 to 5-tibialis anterior, unstimulated, 6 to lo-tibialis anterior, stimulated fibers, note fiber 6 has fast type light chains (see also 3 gel in Fig. 3); 11-soleus fiber; 12-myosin from the soleus muscle. One-half of fiber 6 was used for PPi gel electrophoresis (see 3 gel in Fig. 3); arrows indicate fast light chains in the stimulated fibers.
The slow myosin light chains-which are trace elements in the control tibialis anterior (see overloaded gel 3 in Fig. 2) corresponding to the small amount of slow fibers in this muscle-were greatly increased (see gels 4 and 5 in Fig. 2), and there was a decrease in the fast-type light chains, particularly LC2 and LC3. The fact that the quantity of the fast LC2 was far less than the sum of the fast LCr + LCs-in accord with previous observations that transformation affects different light chains to different extents (17)-suggests that some myosin molecules contained a mixture of fast and slow light chains, or were incomplete in that LC2 might be missing. The two types of myosin light chains occurred in the same fiber as shown by SDS-gel electrophoresis of isolated single fibers (Fig. 3), which indicates a gradual transformation of the fiber from the original fast type to the slow type as a result of stimulation. Electrophoresis under nondissociating conditions (PPi gel) showed in most fibers the essentially total disappearance of the fast-type isozymes and the appearance of the slow-type isozyme and
SRETER ET AL.
FIG. 4. Nondenaturing (PPi) gel electrophoretograms of stimulated and control tibialis anterior single fibers, coelectrophoresed with soleus fibers (1 and 2), and fibers from stimulated tibialis anterior (3 to 6). Polyacrylamide gels (3.6%) were prepared according the method of Hoh (4). Myosin loads are about 1 to 2 pg. Electrophoresis was carried out at a constant voltage of 40 V for 24 h with buffer recirculating at about 80 ml/min. Staining and destaining was carried out according to Lowey et al. (8). I-coelectrophoresis of soleus fiber (uppermost band) and a nonoxidative fast fiber from unstimulated muscle; 2-coelectrophoresis of soleus fiber (uppermost band) and an oxidative fast fiber from unstimulated muscle; 3-fast fiber from stimulated tibialis anterior that has retained fast characteristics. One-half of this fiber was used for SDS-gel electrophoresis (see gel 6 in Fig. 2); 4-fiber containing single isozyme with migration velocity as that of the soleus; 5 and 6-fibers with additional bands.
a new band whose mobility differed from either the slow or the fast isozymes (Fig. 4). The new band was consistent with the presence of an incomplete (lack of a light chain, see above) or hybrid myosin (one type of heavy chain combined with the other type of light chain or two different heavy chains within a dimer) during the transformation period. DISCUSSION It seems to be generally accepted that transformation of one muscle type to another by indirect electrical stimulation of a muscle requires a stimulus pattern resembling that which is supposed to reach the muscle via its own nerve under physiologic conditions. The well-documented transformation of fast muslces into slow ones by chronic electrical stimulation at 10 Hz resulting in changes of contractile characteristics, myosin ATPase, and light-chain pattern, Ca’+ uptake of the sarcoplasmic reticulum, and shifts in the metabolic enzyme pattern fits this concept. The reverse experiment, viz., the transformation of a slow muscle to a fast one by stimuli at higher frequencies, e.g., 100 Hz, has been attempted on denervated muscle, but it has not produced unequivocal results (5, 7). The present work clearly
shows that the fast muscle can be transformed to a slow one by electrical impulses via the intact nerve, in a stimulus pattern physiologically closer to a fast muscle than a slow muscle (2, 3). Our results suggest that the transformation of muscle types is related to the total activity imposed on the muscle rather than to the pattern of stimulation. Not only stimulation at a low frequency similar to that of tonic motoneurons but also stimulus trains at a higher frequency resembling the discharge frequency from the phasic neurons caused a shift from fastto slow-type fibers. It should be noted that this fast to slow transformation was achieved in both cases by a nonphysiological prolonged activation, which was maintained for weeks without intermission. On the other hand, transformation of a slow-twitch muscles to the fast-twitch type can be brought about by inactivity, e.g., cordotomy or chronic immobilization (5). The importance of the amount of activity imposed on the muscle in determining the direction and degree of transformation is supported by other evidence. Intermittent application of IO-Hz stimuli (8 h daily for 7 weeks) shifted the enzyme pattern from oxidative to glycolytic (13) and led to a type 2B to 2A fiber transformation (11). This suggests that the type 2B to 2A transformation may be an intermediate stage in the overall fast to slow transformation. In fact, it seems that a type 2B to 2A transformation preceeds type 2 to type 1 transformation in early stages of continuous loHz stimulation (unpublished). REFERENCES 1. BROOKE, M. H., AND K. K. KAISER. 1970. Muscle fiber types: how many and what kind? Arch. Neural. 23: 369-379. 2. DENNY-BROWN, D. 1929. On the nature of postural reflexes. Proc. R. Sot. (Biol.). 104: 252-301. 3.
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ECCLES, J. C., R. M. ECCLES, AND A. LUNDBERG. 1958. The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol.(London) 142: 255291. HOH, J. F. Y., P. A. MCGRATH, AND R. T. WHITE. 1976. Electrophoretic analysis of multiple forms of myosin in fast-twitch and slow-twitch muscles of the chick. B&hem. J. 157: 87-95. JOLESZ, F., F. A. SRETER, K. MABUCHI, K. PINTFIR, AND J. GERGELY. 1981. Effect of various forms of hype- and inactivity on slow muscle. Pages 57-68 in G. MARECHAL, F. GUBA, AND 0. TAKACS, Ms., Mechanism of Muscle Adaptation lo Functional Requirements, Vol. 24, Advances in Physiological Sciences. Pergamon, Oxford/New York. LAEMMLI, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685. LIMO, T., R. H. WESTGMRD, ANDL. ENGEBRETSEN. 1980. Different stimulation patterns affect contractile properties of denervated rat soleus muscles. Pages 297-309 in D. PET~E, Ed., Plasriciiy of Muscle. de Gruyter, Berlin/New York. LOWEY, S., P. A. BENFIELD, L. SILBERSTEIN, AND L. M. LANG. 1979. Distribution of light chains in fast skeletal myosin. Nature (London) 282: 522-524.
9. MABUCHI, K., AND F. A. SRETER. 1978. Use of cryostat sections for measurement of Caz+-uptake by sarcoplasmic reticulum. Annl. Biochem. 86: 733-742. 10. MABUCHI, K., AND F. A., SRETER. 1980. Actomyosin ATPase II. Fiber typing by histochemical ATPase reaction. Muscle & Nerve 3: 233-239. 11. MABUCHI, K., K. PINTER, P. ALLEN, J. GERGELY, AND F. A. SRETER. 1981. Characteristics of human and rabbit single fibers. Pages 69-78 in G. MARECHAL, F. GUBA, ANDO. TAKACS, Eds., Mechanism of Muscle Adaptation to Functional Requirements, Vol 24, Advances in Physiological Sciences. Pergamon, Oxford/New York. 12. NOVIKOFF, A. B., W. Y. SHIN, AND J. DRUCKER. 1961. Mitochondrial localization of oxidative enzymes: staining results with two tetrazolium salts. J. Biophys. Biochem. Cytol. 9: 47-61. 13. PETTE, D., W. MULLER, E. LEISNER, AND G. VRBOVA. 1976. Time dependent effects on contractile properties, fibre population, myosin light chains and enzymes of energy metabolism in intermittently and continuously stimulated fast twitch muscles of the rabbit. PfUgers Arch. 364: 103-l 12. 14. ROMANUL, F. C. A., F. A. SRETER, S. SALMONS, AND J. GERGELY. 1974. The effect of changed pattern of activity on histochemical characteristics of muscle fibers. Pages 344-348 in A. T. MILHORAT, Ed., Exploratory Concepts in Muscular Dystrophy, II Int. Congr. Series No. 333. Excerpta Medica, Amsterdam. 15. SALMONS, S., AND G. VRBOVA. 1969. The influence of activity on some contractile characteristics of mammalian fast and slow muscles. J. Physiol. (London) 201: 535-549. 16. SRETER, F. A., J. GERGELY, S. SALMONS, AND F. C. A. ROMANUL. 1973. Synthesis by fast muscle of myosin light chains characteristic of slow muscle in response to longterm stimulation. Nature New Biol. 241: 17-19. 17. SRETER, F. A., F. C. A. ROMANUL, S. SALMONS, AND J. GERGELY. 1974. The effect of changed activity pattern on some biochemical characteristics of muscle. Pages 344-348 in A. T. MILHORAT, Ed., Exploratory Concepts in Muscular Dystrophy, II Int. Congr. Series No. 333. Excerpta Medica. Amsterdam.