Appearance in Slow Muscle Sarcolemma of Specializations Characteristic of Fast Muscle after Reinnervation by a Fast Muscle Nerve

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1 EXPERIMENTAL NEUROLOGY 58, (1978) Appearance in Slow Muscle Sarcolemma of Specializations Characteristic of Fast Muscle after Reinnervation by a Fast Muscle Nerve MAKK H. ELLISMAN, MICHAEL H. BKOOKE, KENNETH K. KAISER, AND JOHN E. RASH l Departmeut of Molecular, Cellular attd Develojmetttal Biology, University of Colorado, Boulder, Colorado; Muscular Dystrophy Research Cetztcr, Washingtow Uxiversity Medical School, St. Louis Missouri; afld Departme& of Pharmacology afrd Experimental Therapeutics, University of Maryland Medical, School, Baltimore, Marylatld Received Jzmc 21, 1977 We utilized quantitative freeze-fracture electron microscopy to study the plasticity of orthogonal clusters of 60-A particles (the square array ) found in the sarcolemma of fast-twitch muscle. The membrane macromolecular composition of normally slow-twitch rat soleus muscle was examined 1 year after surgical reinnervation by the nerve from fast-twitch extensor digitorum longus muscles. The isometric contraction times and histochemical profiles were monitored and it was confirmed that conversion of fiber types had occurred. The sarcolemma of the switched fast soleus developed square arrays of 60-A particles characteristic of fast-twitch muscle whereas the sarcolemma of the contralateral control, the slow soleus, contained only random particles. Square array density per square micrometer in cross-reinnervated fast soleus fibers resembled that of normal fast extensor digitorum longus muscles and varied as a function of distance from the neuromuscular junction. This experiment demonstrates that the appearance of these unusual clusters of 60-8, particles is neuronally regulated. We further suggest that these macromolecules are under the influence of the same subtle aspects of innervation that regulate the differentiation of myosin adenosine triphosphatase and thereby the contractile behaviors of fast- and slow-twitch muscle. The function of the sarcolemmal square array is unknown ; however, a correlation with a membrane property that is more highly developed in fast-twitch muscle is to be expected. Abbreviations : ATPase-adenosine triphosphatase ; EDL-extensor digitorum longus ; SOL-soleus. 1 Dr. Ellisman was in Boulder, Drs. Brooke and Kaiser are in St. Louis, and Dr. Rash is in Baltimore. Dr. Ellisman s present address is Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA /78/ $02.00/O 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

2 60 ELLlSMAN ET AL. INTRODUCTION The membranes of fast- and slow-twitch muscles of the rat differ in their macromolecular architecture ( 13) and biochemical composition (14, 15). In freeze-fracture, fast-twitch muscle membranes are distinguished by their unusual complement of orthogonal aggregates of 60-A particles, the so-called square array (21) whereas slow-twitch muscle membranes rarely exhibit this specialization (12, 13). Differentiation of biochemical properties of the sarcolemma includes variations between gel staining for Na+-K+ adenosine triphosphatase ( ATPase) bands (15) and adenylate cyclase (14). It was demonstrated that the contractile properties of these muscles are influenced by properties of the innervating nerve (6, 7). These differences in contractile properties have been correlated with differences in the type of myosin ATPase (1). Further, experimental cross-reinnervation of rat soleus (SOL) (slow-twitch) and extensor digitorum longus (EDL) (fast-twitch) muscles results in a reversal of both the contractile properties (7, 8) and the species of myosin ATPase found (2, 9, 18). The experiments reported here were designed to determine if the membrane specializations of fast-twitch muscle (the orthogonal arrays) would appear in slow-twitch muscle membranes upon cross-reinnervation by the nerve which once innervated a fast-twitch muscle. MATERIALS AND METHODS Nerve-Switch Operations. Operative reinnervation of the soleus with the nerve from the EDL was carried out on four S-week-old male albino rats under aseptic conditions ; the anesthetic agent was pentobarbital sodium, 50 mg/kg body weight, injected intraperitoneally. Soleus and EDL nerves were exposed by reflecting the neighboring muscles and were transected near the point of entry to the muscle. The EDL nerve was partially separated from the common peroneal nerve to obtain sufficient length to reach the soleus muscle. The EDL nerve and the soleus muscle were then united by suturing the nerve stump to the point of previous nerve entry on the muscle (edlx SOL) _ In an additional group of four animals, the soleus nerve was similarly severed but sutured back onto the soleus muscle, so as to create a self-reinnervation control (sol X SOL). Dissection of iwu.scles for Experintents. Nine to twelve months later the animals were anesthetized with pentobarbital sodium as in the initial operation. The EDL nerve-innervating soleus muscle was dissected by exposing the common peroneal nerve throughout its length and transecting it near the point where it arises from the sciatic nerve; all branches of the common peroneal nerve were cut except the EDL nerve to the soleus muscle. The contralateral control soleus nerve was prepared in a similar way by exposing the sciatic nerve, transecting it near the sciatic notch, and cutting all

3 MUSCLE MEMBKANE PLASTlCITY 61 nerves to muscles except the soleus nerve to soleus muscle. To ensure that the reinnervation of the soleus had resulted in an increase in the speed of contraction, we measured and compared the isometric twitch contraction times of EDL nerve-innervated soleus muscle (edlx SOL), soleus nerveinnervating soleus muscle (sol-sol), and EDL nerve-innervating EDL muscle (edl-edl). The muscles were dissected free from surrounding muscles and prepared for in situ recording of isometric contractions in the following manner. Recording of Contraction Tim,es. After suspending the animal in a prone position, flaps of skin over each of the dissected limbs were tied up to create baths into which flowed oxygenated Krebs-Ringer s solution (with the following composition in millimoles per liter: NaCl, 135 ; KCl, 5.0; MgClz, 1.0; CaC12, 2.0; NaHCO& 15.0; Na2HP04, 1.0; glucose, 11.0) maintained at 35 to 37 C. Isometric contractions of each muscle were recorded with the proximal tendon clamped securely to a rigid frame and the distal tendon attached directly to a strain gauge (Statham ). Signals were recorded directly with an oscilloscope camera. Prior to stimulation, muscle resting tension was adjusted to 2 gs. The muscles were stimulated by impulses delivered to the isolated nerve from a Grass SD9 stimulator. Supramaximal stimuli, 0.2-ms duration, were delivered at a low frequency (one cycle per 10 s) to avoid staircase effects and post-tetanic potentiation. Preparation of Tissues for Electron Microscopy and Histochemistry. Subsequent to the recording of contraction times, the muscles were fixed and prepared for electron microscopy. Details of the electron microscopic procedures are found in Rash and Ellisman (21). An explanation of the present data sampling procedures is detailed in Ellisman et al. (13). Muscle was prepared for histochemical reactions by freezing in isopentane, cooled to -170 C in liquid nitrogen, and sectioned at lo-pm thickness in the cryostat. In addition to the routine trichrome and hematoxylin and eosin stains, NADH-tetrazolium reductase and ATPase reactions, including those modified by preincubation, were carried out. The procedures are standard and are outlined in Dubowitz and Brooke (11). OBSERVATIONS Isometric Contraction Times. Figure 1 shows representative contraction times of experimental (edlx SOL) and control contralateral (sol-sol and edl-edl) muscles. The contraction time of normal soleus (sol-sol) is the solid line, that of normal EDL (edl-edl) is the dashed line, and that of the soleus reinnervated with the nerve from EDL (edlx SOL) is the dotted line. From this superpositioning of measurements it is readily apparent that the contraction time of edlxsol is nearly that of normal fast edl-edl. Isometric twitch contraction times were the only physiologic properties

4 62 ELLISMAN ET AL ,,,,,,,,,,,,,,, IO msec FIG. 1. A comparison of tension: time curves for isometric contractions of normal soleus (sol-sol, solid line), normal EDL (edl-edl, dashed line), and soleus reinnervated with the nerve from EDL (edlxsol, dotted line). These isometric twitch responses were recorded just prior to fixation 300 to 350 days postoperative. monitored because they constitute a relatively simple and accurate measure of the success of the surgical manipulation. Muscle Histochewzistry. The routine ATPase reaction, incubated at ph 9.4, characterizes two fiber types. Type 1 fibers usually correspond to physiologically slow fibers and the majority of fibers in the normal soleus are of this type. The pattern of the ATPase reaction may be changed by preincubating the tissue at various phs (3, 16). Several different systems of fiber type nomenclature are currently in use [see (4) 1. The present paper will use a system of classification based on the ATPase characteristics. Type 1 fibers are relatively lighter stained with the routine ATPase procedure. Type 2 fibers are more densely stained and may be subdivided, on the basis of their ph sensitivity, into 2A, B, and C. Type 2C fibers correspond to histochemically undifferentiated fibers and usually disappear with increasing age; the exception to this is seen in the soleus. Type 2A fibers probably correspond to the fast oxidative glycolytic fibers in the specific muscles discussed in this paper. Type 2B fibers are fast glycolytic fibers. Although the fiber typing is controversial, for the present purpose the histochemical reactions are used as convenient indicators of fiber types. The soleus muscle in the rat consists chiefly of type 1 fibers (Fig. 2a), with a few 2A and undifferentiated (2C) fibers. The EDL consists primarily of 2B and 2A fibers with a few type 1 fibers. It has been shown repeatedly that cross innervation and reinnervation can change the histochemical fiber type of the muscle and also result in a phenomenon known as type grouping, in which clumps of one fiber type are seen next to clumps of another type (5, 17,20,22).

5 MUSCLE MEMBRANE PLASTICITY 63 Transformation of the histochemical fiber type and fiber type grouping which indicated successful reinnervation was observed in our preparations (Fig. 2b). In the cross-reinnervated soleus the majority of fibers was type 2B (an average of 70% with lesser numbers of types 1 and 2A fibers). The conversion of this predominantly type 1 muscle to a predominantly type 2 form may be ascribed to the influence of the nerve. The reinnervation of soleus by a fast nerve is therefore confirmed by the histochemical picture as well as the physiological tests described in the previous section. Mevzbrane Ultrastructye. Examination of freeze-fracture replicas from cross-reinnervated and control muscles reveals that the soleus muscle reinnervated by an EDL nerve acquired an abundance of square arrays not normally characteristic of its membranes (Fig. 3). For each of the soleus muscles (experimental and the two controls), three sarcolemmal regions representing measured distances from the motor end plate were sampled for array density : (i) tissue samples containing neuromuscular junctions, FIG. 2. Cross sections from the soleus muscles stained by modified ATPase reaction following preincubation at ph 4.5. a-normal soleus (sol-sol) demonstrating a homogeneous population of type 1 fibers. b-cross-reinnervated soleus (edlx SOI,) demonstrating homogeneous clumps of types 1, ZA, and 2B fibers (fiber type grouping). This cross-innervated muscle has been converted from a predominantly type 1 muscle to a predominantly type 2 muscle. X 80.

6 64 ELLISMAN ET AL. CROSS- INNERVATED SOLEUS (fast twitch I Ng 30 \ l CT E 20 i &.. : :.... i:... & :..... NMJ contolning somp. IQ-lmm samples I each l represents 5#m2 of micrographs from PF repltcos counted for square arrays1 40- NORMAL SOLEUS (slow twitch] 30 - E?!A e 20-m 2 e 9 :: IO-... :. FIG. 3. Quantitative comparison of sarcolemmal square array densities as a function of distance from the motor end plates. Cross-innervated soleus (edlxsol) above, normal control soleus (sol-sol) and self-innervated soleus (solxsol) below. (See text for additional detail.) NMJ-Neuromuscular junction; PF-protoplasmic fracture face. (ii) samples 0.5 to 1 mm from the junctions, and (iii) samples 2 to 4 mm from the junction. These three distances are represented as the three separate clusters of dots along the horizontal axis in Fig. 3. Each dot represents five per square micrometer of sarcolemmal protoplasmic leaflets counted for square arrays. The density of arrays observed at each distance is then represented by the scatter of dots on the vertical axis. Two observations are evident from the data. First, edlx SOL has an abundance of square arrays not seen in the controls, sol-sol and sol x SOL. Second, the density of arrays in the sarcolemma of edlxsol varies as a function of distance from the neuromuscular junction, as was demonstrated for normal EDL (13). Figure 4a is a representative micrograph depicting a high density of square arrays from edl~s0l. Figure 4b represents the contralateral control (sol-sol) with no arrays.

7 MUSCLE MEMBRANE PLASTICITY 65 FIG. 4. a-cross-reinnervamted (edlxsol) sarcolemma 2 to 4 mm from an end plate. Note the numerous orthogonal clusters of 60-a particles ( square arrays ). b-normal control contralateral soleus (sol-sol) sarcolemma 2 to 4 mm from an end plate with no square arrays apparent. X 120,000. DISCUSSION This report describes the appearance in slow-twitch muscle (soleus) membranes of macromolecules normally characteristic of fast-twitch muscle (13). This response occurs following cross reinnervation by phasically active motor neurons. Simple denervation alone does not induce the appearance of square arrays in soleus membranes or their disappearance from membranes of EDL muscle (Ellisman and Rash, 1977, Brain lies., in press). Thus, it appears that molecular specializations of postsynaptic membranes are influenced by factors distinguishing these two types of innervation from one another. Experiments designed to determine whether these influences may result from trophic interactions, pattern of presynaptic firing, pattern of postsynaptic depolarization, or other factors resulting from neuronal differences would help clarify the nature of the neuronal effect on the postsynaptic membrane. The appearance of orthogonal arrays in soleus membranes may result from synthesis of new membrane proteins by the soleus fibers. Alternatively the recognizable aggregates may simply aggregate from a preexisting pool of subunits in response to the differing pattern of activation or other spe-

8 66 ELLISMAN ET AL. cific trophic factors. We are currently undertaking experiments designed to distinguish between these possibilities. Our preliminary observations indicate that the array is a labile structure, disaggregating under certain conditions such as anoxia. Barbiturate anesthesia, as was used in these studies, seems to stabilize or increase the aggregation of subunits in EDL muscle but does not stimulate the appearance of arrays in soleus muscle (Ellisman, in preparation). In this regard it is noteworthy that preliminary biochemical analysis of partially purified sarcolemma of EDL and soleus (Everhart, Rash and Ellisman, in progress) reveals two proteins, M 117,- 000 and 130,000, present in large amounts in EDL, but virtually absent in soleus. Whether either or both of these proteins can be correlated with the square array has not been determined. We do, however, wish to call attention to striking differences in membrane composition. The identity of the square array remains enigmatic. Similar arrays have been observed in many specialized tissues [for a recent summary see (13) 1. Recent differential biochemical analysis of fast and slow sarcolemmas revealed differences in gel staining of Na-K ATPase bands ( 15) and activity of adenylate cyclase (14). A differentiation of myosin ATPase kinetics between fast and slow muscle is established and considered the basis for the difference in speed of shortening (10). The neural regulation of myosin ATPase phenotype is also accepted and widely explored (19). Physiological functions served by the differences in muscle membrane properties may have a possible obligatory interrelationship with the muscle s contractile properties. In addition, the manner in which these membrane properties are influenced by aspects of innervation are now subject to experimental manipulation. REFERENCES 1. BARANY, M ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50 : BARANY, M., AND R. I. CLOSE The transformation of myosin in cross-innervated rat muscles. J. Physiol. (London) 213 : BROOKE, M. H., AND K. K. KAISER Some comments on the histochemical characterization of muscle adenosine triphosphatase. J. Histochem. Cytochem 17 : BROOKE, M. H., AND K. K. KAISER Muscle fiber types: How many and what kind? Arch. Neurol. 23 : BROOKE, M. H., E. WILLIAMSON, AND K. K. KAISER The behavior of the four principal muscle fiber types in the developing rat and in reinnervated muscle. Arch. Neurol. 25 : BULLER, A. J., J. C. ECCLES, AND R. M. ECCLES Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. (London) 150 : BULLER, A. J., J. C. ECCLES, AND R. M. ECCLES Interactions between motoneurons and muscles in respect of the characteristic speeds of their response. J. Physiol. (Lomion) 150 :

9 MUSCLE MEMBRANE PLASTICITY BULLER, A. J., C. J. C. KEAN, AND K. W. RANATUNGA Force-velocity characteristics of cat fast and slow-twitch skeletal muscle following cross-innervation. J. Physiol. (Lofzdon) 213 : BULLER, A. J., W. F. H. M. MOMMAERTS, AND K. SERAYDARIAN Enzymatic properties of myosin in fast and slow twitch muscles of the cat following crossinnervation. J. Physiol. (Loxdon) 205 : CLOSE, R. I Dynamic properties of mammalian skeletal muscles. Pkysiol. REV. 52 : DUBO\I ITZ, V., AND M. H. BROOME Muscle Biopsy: d Modem A4pproacll. W. B. Saunders, London/Philadelphia/Toronto. 12. ELLISMAN, M. H., J. E. RASH, L. A. STAEHELIN, AND K. R. PORTER Freeze-fracture comparisons of the neuromuscular junction and postjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers. J. Cr2l Biol. 63: 93a. 13. ELLISMAN, M. H., J. E. RASH, L. A. STAEHELIN, AND K. R. PORTER Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and non-junctional sarcolemmas of mammalian fast and and slow twitch muscle fibers, J. Cell Biol. 68 : FESTOFF, B. W., K. L OLIVER, AND N. B. REDDY In vitro studies of skeletal muscle membranes: Adenylate cyclase of fast and slow twitch muscle and the effects of denervation. J. Mewbr. Biol. 32 : FESTOFF, B., K. L. OLIVER, AND N. B. REDDY I)t vitro studies of skeletal muscle membranes: Effects of denervation on the macromolecular components of cation transport in red and white skeletal muscle. J. Mnrzbr. Biol. 32 : GUTH, L., AND F. J. SAMAHA Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. Exp. Nczwol. 25: GUTH, L., P. K. WATSON, AND W. C. BROWN Effects of cross-reinnervation on some chemical properties of red and white muscles of rat and cat. E.rp. Ncwol. 20 : HOH, J. F. Y Neural regulation of myosin types in muscle. Proc. <4zrst. Plq.siol. Plrarncacol. Sot. 3 : HOH, J. F. Y Neural regulation of muscle activation. Exi). Newel. 45: KARPATI, G., AND W. K. ENGEL Transformation of the histochemical profile of skeletal muscle by foreign innervation. Natwr (Londou) 215 : RASH, J. E., AND M. H. ELLIS&IAN Studies of excitable membranes, I. Macromolecular specializations of the neuromuscular junction and the nonjunctional sarcolemma. J. Cell. Biol. 63 : RORIANUL, F. C. A., AND J. P. VAN DER MEULEN Reversal of the enzyme profile of muscle fibers in fast and slow muscles by cross-innervation. Nutzrrc (Londou) 212 :