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gastrocnemius muscle were found to be well maintained during a 180minute perfusion. An example of the hormone responsiveness of the hemicorpus preparation is illustrated by the data shown in Fig. 4. In these experiments the effects of time of perfusion and insulin on the rate of protein synthesis in psoas muscle were studied. The rate of protein synthesis was linear during the first hour of perfusion, but declined sharply during the second and third hours (left panel, Fig. 4). The decline in the rate of protein synthesis was associated with increased numbers of ribosomal subunits and decreased numbers of polysomes (center and right panels, Fig. 4). The number of polysomes and ribosomal subunits is influenced by rates of initiation and elongation of peptide chains. Increased numbers of subunits and decreased numbers of polysomes result when initiation is retarded relative to elongation. 1~ These findings indicated that the decline in the rate of protein synthesis was due to a block in peptide-chain initiation which had developed during perfusion, perhaps as a result of depletion of hormones and substrates. Addition of insulin prevented development of the block in peptide-chain initiation as evidenced by maintenance of the initial rate of protein synthesis and in vivo levels of ribosomal aggregation. Other experiments indicated that glucose and fatty acid metabolism of the hemicorpus were also responsive to the in vitro addition of insulin. 14 The maintenance of the tissues of the hemicorpus in a good physiological state for perfusion periods up to 3 hours and the responsiveness of metabolic pathways to in vitro addition of hormone indicate the suitability of the preparation for studies of the metabolism of skeletal muscle. la H. E. Morgan, L. S. Jefferson, E. B. Wolpert, and D. E. Rannels, J. Biol. Chem. 246, 2163 (1971). 1~L. S. Jefferson, J. O. Koehler, and H. E. Morgan, Proc. Nat. Acad. Sci. U~S. 69, 816 (1972).
[10] I n V i t r o P r e p a r a t i o n s o f t h e D i a p h r a g m and Other Skeletal Muscles By ALFaED L. GOLDBERG,SUSAN B. MARTEL, and MARTIN J. KUSHMERICK Isolated mammalian muscles incubated in vitro offer numerous advantages for controlled investigations of muscle metabolism and neuromuscular physiology and pharmacology. This article will review certain prepa-
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r a t i o n s of skeletal muscle t h a t h a v e p r o v e d to be useful for biochemical a n d physiological studies in vitro. T h e m a j o r difficulty in m a i n t a i n i n g tissues in v i t r o has been e n s u r i n g t h a t a d e q u a t e a m o u n t s of n u t r i e n t s reach the i n d i v i d u a l cells b y diffusion in the absence of a c i r c u l a t o r y system. As a result, studies with i n c u b a t e d muscles have been restricted to especially t h i n muscles, o b t a i n e d from s m a l l rodents, t T h e v i a b i l i t y of such muscles in v i t r o g e n e r a l l y exceeds t h a t of slices from other m a m m a l i a n tissues, such as liver, k i d n e y , spleen, or brain. P e r f u s i o n of a specific muscle or whole h i n d l i m b s has also been used with success b y several laboratories2; however, the t e c h n i q u e s i n v o l v e d in isolation a n d i n c u b a t i o n of muscles in vitro are far simpler t h a n those r e q u i r e d for tissue perfusion. I n this discussion, special e m p h a s i s will be placed on the r a t diap h r a g m which has been the most f r e q u e n t l y s t u d i e d muscle p r e p a r a t i o n since it was first i n t r o d u c e d b y M e y e r h o f a n d H i m w i c h 3 n e a r l y fifty y e a r s ago. T h e i n c u b a t e d d i a p h r a g m was p o p u l a r i z e d p r i m a r i l y by the work of G c m m i l P a n d K i p n i s a n d Cori, 5 who d e m o n s t r a t e d t h a t it was a c o n v e n i e n t p r e p a r a t i o n for studies of h o r m o n a l control of c a r b o h y d r a t e m e t a b o l i s m a n d t r a n s p o r t . T h e m a j o r a d v a n t a g e s of the d i a p h r a g m are 'As a result of diffusion and metabolism, cells within an incubated muscle may be exposed to concentrations of nutrients different from those found in the incubation medium (A. V. Hill, "Trails and Trials in Physiology," p. 208, Williams & Wilkins, Baltimore, Maryland 1965). This difference will depend on the geometry of the tissue, the distance of a specific cell from the nearest surface, and the rate of utilization or release of substances from the cells. Thus in a flat tissue which consumps O2 at a rate A (measured in units of cm30~ STP/cm 3 tissue per minute), the distance X from the surface at which the O.~ concentration falls to zero is given by X = ( 2 K Y / A ) '/2, where K is Krogh's diffusion constant and Y is the O~ tension in the incubation medium measured as the partial pressure fraction. In muscle, K for O: is 1.66 × 10-~ cm~ per minute at 37°. For the rat diaphragm, basal O~ consumption rate at 37 ° is 0.5/zmole per gram per minute, and thus the critical depth into the muscle at which O~ tension falls to zero is 0.54 mm for incubations in 100% O~. For cylindrical muscles the appropriate formula is R = 2 ( K Y / A ) 1/0". Assuming similar rates of oxygen consumption for such muscles, the critical radius for in vitro incubations is about 0.77 mm. Since the Q10 for oxygen consumption is about 2.5 and for Krogh's constant K is about 1.1, it is clear that thicker muscles should be used only at temperatures below 37 ° to assure adequate oxygenation of all fibers; thus at 20 °, the critical distances are approximately twice that at 37 ° . 2L. S. Jefferson, this volume [91; J. T. Stull and S. E. Mayer, this volume [8]. 2, N. B. Ruderman, C. R. S. Houghton, and R. Hems, Biochem. J. 124, 639 (1971). ~O. Meyerhof and I-I. E. Himwieh, Pfluegers Arch. Gesamte Physiol. 205, 415 (1924). C. L. Gemmill, Bull. Johns Hopkins Hosp. 68, 329 (1941). D. M. Kipnis and C. F. Cori, J. Biol. Chem. 224, 681 (1957).
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I A
B
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't c
FIG. 1. Drawings of rat diaphragm (A), extensor digitorum longus (B), and soleus (C), after L. T. Potter [J. Physiol. (London) 206, 145 (1970)] and R. A. Close [Physiol. Rev. 52, 129 (1972)]. Darkened areas indicate tendon. For mature rats (200 g) the calibration line adjacent to each muscle indicates 1 cm; in younger rats routinely used in our labs (50-70 g) the calibration is approximately 0.5 cm. The diaphragm is drawn as viewed from the thoracic cavity with the dorsal segment on top. Lines are drawn radially to indicate the orientation of the muscle fibers from the central tendon. The ribs have been removed. Note the more diffuse arrangement of nerve terminals (dots) in the right hemidiaphragm. In drawings of the extensor digitorum longus (B) and soleus (C) the right member of the pair represents a lateral view of the muscle; on the left are anteroposterior views. The distal tendons are uppermost in each case. Note that the soleus is thicker than the extensor digitorum longus. its rapid dissection and excellent viability in vitro. In addition, since the diaphragm can be readily dissected in combination with a considerable length of phrenic nerve, this preparation has been used widely by neurophysiologists and neurochemists. Various other preparations of mammalian skeletal muscle have been introduced. One major motivation for identifying other muscles that are convenient for study in vitro is that mammalian muscles are composed of at least three distinguishable types of fibers which differ in their physiological, histochemical, and ultrastructural properties2 Different muscles can v a r y in the relative proportions of the three fiber types and therefore can differ appreciably in their metabolic, endocrinological, and pharmacological responses. The Rat Diaphragm
Anatomy. In rats and mice, the diaphragm is an unusually thin sheet of muscle fibers that in young animals is even translucent. The muscle is composed of three distinct regions (Fig. 1) : two larger lateral portions, which are generally referred to as "hemidiaphragms," and a smaller dor6 R. A. Close, Physiol. Rev. 52, 129 (1972).
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sal segment adjacent to the spinal cord through which pass the esophagus, aorta, and vena eava. These subdivisions can be readily separated without damaging the fibers by cutting along the Y-shaped central tendon that delineates these three muscular segments. Within each hemidiaphragm, the muscle fibers run in a radial direction from the rib cage toward the central tendon. The neuromuscular junctions and the intradiaphragmatic portions of the phrenie nerve are localized in a well-defined thin band in the center of the muscle fibers that can be recognized with the naked eye (Fig. 1). In the diaphragm, red fibers predominate, but white and intermediate fibers each compose 20% of the total. Most investigators have discarded the smaller triangular segment of muscle and compared the responses of the contralateral hemidiaphragms using one as the control. It is also possible to cut the hemidiaphragm parallel to the muscle fibers so as to produce several segments of tissue from the same diaphragm. For most metabolic investigations in this laboratory, we routinely bisect the hemidiaphragm and compare the four "quarter diaphragms" under different experimental conditions. Thus far, the behavior of the "quarter diaphragm" has proved indistinguishable from that of the hemidiaphragm. Comparisons between segments from the same muscle offer obvious statistical advantages in reducing biologieal variability. Such internally controlled data are more difficult to obtain with other muscle preparations and impossible in perfused muscles. However, there are certain anatomical differences between the right and left hemidiaphragms. For example, the left hemidiaphragm with nerve attached has been used in most electrophysiological and biochemical studies of the neuromuscular junctions, because, on the left side, the region containing the nerve endings and neuromuscular junctions is more sharply delineated than on the right (Fig. 1). ~ In addition, the left phrenie nerve is simpler to dissect (or to section surgically) in the thorax than the right nerve. It thus seems advisable to cheek whether the behaviors of the contralateral hemidiaphragms are identical, at the outset of any investigation. Dissection o] the Diaphragm. In order to avoid problems of diffusion of nutrients into the incubated muscle, 1 it appears advisable to use rats weighing less than 100 g for most experiments. The best preparations of diaphragms from adult rats (280-320 g) lose most of their ATP and hexose phosphate intermediates upon incubationS; these findings contrast with those obtained with tissues from young rats (see below). For rectabolic studies, we routinely use 60-80 g rats (3-4 weeks of age), in which the wet weight of the hemidiaphragms ranges between 40 and 50 rag. L. T. Potter, J. Physiol. (London) 206, 145 (1970). K. A. Rookledge, Biochem. J. 125, 93 (1971).
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Such animals are rapidly growing, which complicates the planning of experiments. Obviously such rats are not appropriate for certain investigations (e.g., experiments on aging), and mouse diaphragm appears more appropriate for studies requiring adult tissues. For rapid removal of the diaphragm from small rats or mice, cervical dislocation by pressure with a rod behind the neck has proved to be a simple and rapid method for sacrificing the animal. Many labs have successfully employed decapitation or stunning by a blow to the head. The latter methods are not advised when diaphragm and phrenic nerve are to be removed together, since they can injure the nerve or cause hemorrhage and blood clotting within the thoracic cavity. For such dissections, anesthesia with ether or barbiturates appears preferable (e.g., 40-50 mg of pentobarbital per kilogram of rat weight with supplementary 10 mg doses as necessary). The diaphragm can be quickly excised along with a narrow ring of ribs to which the muscle is attached. After cutting through the skin on the ventral surface, the pectoral and abdominal muscles are trimmed from the rib cage. The lower edge of the diaphragm is next freed by cutting the several ligamentous attachments of the liver. The spinal column, esophagus, vena cava, and aorta are then severed below the diaphragm. The pleural cavity is entered by cutting through the sternum slightly above the xiphoid process, and the incision is extended on both sides to the spinal column. Finally the pericardial membranes and spinal column are severed above the diaphragm. The entire diaphragm with the supporting ribs and intercostal muscles is then transferred to a petri dish containing oxygenated buffer at room temperature. With practice, the time required from sacrifice to transfer to the petri dish can be shortened to less than 1 minute. The actual period of ischemia of the muscle is probably much shorter since the circulation remains intact until the aorta and vena cava are severed. The preparation in the petri dish is trimmed by removing the xiphoid process, spine, and dorsal segment of diaphragm. The central tendon is then bisected to yield two intact muscle masses. If necessary, the intercostal muscles and ribs are further trimmed to minimize the amount of tissue extraneous to the diaphragm. Since these associated tissues can weigh 10-50 times the hemidiaphragm, they may affect the composition of the incubation medium either by leaking or utilizing substances. The Diaphragm with and without Ribs
Unfortunately the diaphragm lacks a clearly defined tendon at the costal margins, and thus the investigator is forced either to incubate the
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87
muscle with a portion of the ribs attached or to risk damage to the diaphragm by attempting to trim away the attached remnants of the rib cage. Both preparations have been used successfully for different purposes. For most studies it is advisable to incubate the hemidiaphragm with some ribs attached. As first shown by Kipnis and Cori, 5 such preparations more closely resemble the diaphragm in vivo and are more stable than hemidiaphragm or quarter diaphragm without ribs. The preparation with ribs is recommended for metabolic studies in which the products to be measured remain within the diaphragm, such as transport of substances into the intracellular space or incorporation of precursors into muscle protein, glycogen, fat, or nucleic acids. After incubation the diaphragm must then be cut away from the ribs, blotted, and weighed prior to chemical measurements. Incubation with ribs, however, cannot be used for studies in which the experimenter monitors loss of substances from the medium (such as measurements of 02 consumption) or evaluates metabolic products that might be released by the diaphragm into the surrounding media (such as measurements of transport out of the muscle, or investigation of CO~, lactate, and amino acid production). For such experiments, the diaphragm must be cut away from its supporting structures prior to incubation, since the greater mass of muscle and bone attached will leak materials into the medium and thus contribute to the values measured. Trimming of ribs must be performed with fine scissors as close to the costal border as possible so as to minimize damage to diaphragm and yet avoid the inclusion of intercostal muscles in the incubation. Gentle blotting and weighing of the tissue is advisable prior to incubation because such preparations often swell during the course of incubation. By a variety of criteria, the preparation with ribs more closely approximates the diaphragm within the organism than that with ribs removed, probably because the latter preparation contains appreciable numbers of injured fibers. (Kipnis and CorP have even termed this latter preparation the "cut diaphragm" to distinguish it from the "intact diaphragm" preparation with ribs.) For example, even after 90 minutes of incubation in oxygenated buffer, quarter diaphragms with ribs attached contain amounts of ATP, inorganic phosphate, and phosphorylcreatine similar to muscles removed immediately from the rat (Table I). In such diaphragms, the extracellular space, 5 the permeability to pentoses, 5 and total intracellular amino acids (estimated in ninhydrin-reactive material or fluoresceamine-reactive material) also did not fall significantly even after incubation for 120 minutes. In addition, the preparations with ribs can maintain nearly normal resting potentials for 4 hours, are capable
88
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S K E L E T A L AND SMOOTH MUSCLE
TABLE I COMPOSITION OF I~¢[ETABOLITES IN RAT DIAPHRAGM ~NCUBATED WITH AND WITHOUT RIBS a
Experimental conditions With ribs Initial 90-minuteincubation at 37° Without ribs Initial 90-minuteincubation at 37°
Creatine
Phosphorylcreatine
Total creatine
9.1 _+ 1 9.0_+ 0.2
12.5 + 1.2 10.8 + 0.5
21.6 + 0.5 4.3__ 0.5 19.8_+ 0.5 4.3 +_ 0.4
5.7 __ 1.l 6.7 _+ 0.2
8.9 __ 0.5 5.6_+ 0.5
12.2 ± 0.3 9.0_+ 0.9
21.3 + 0.4 4.0 +_ 0.5 14.7 _+ 1.3 3.5_+ 0.5
6.1 _+ 0.9 6.9 _+ 0.7
ATP
Inorganic phosphate
a Each value is the mean _+ SE of four determinations, each of which involved quarter diaphragms from three animals. Values are expressed as micromoles per gram of blotted wet weight. After freezing of the tissues in isopentane at - 150°C, the metabolites were extracted and analyzed by standard methods [M. J. Kushmerick, R. E. Larson, and R. E. Davies, Proc. Roy. Soc. Ser. B 174, 293 (1969)]. of contraction for up to 12 hours, and incorporate labeled amino acids into protein at constant rates for 4 hours. I n contrast, in diaphragms without ribs attached, the levels of creatine and phosphorylcreatine fall during incubation for 90 minutes. The total ereatine content (i.e., creatine and phosphorylcreatine combined) can be a useful measure of functional muscle mass, 9 and m a y even be a more reliable measure than wet weight, especially if some swelling or damage occurs. This fall in total creatine wouId suggest that some fibers in the muscle were damaged upon removal of the ribs and leaked metabolites into the medium. In support of this conclusion, creatine and phosphorylcreatine were recovered in the medium in amounts equal to those lost from the muscle (Table I I ) . Glucose, insulin, and amino acids at 5 times plasma concentrations did not prevent this leakage of total creatine. Furthermore, the preparation leaks several percent of its protein into medium. Although the diaphragm m a y leak intracellular components during the first half hour after the ribs are trimmed away, this preparation subsequently appears stable by several criteria. After the initial decrease, the concentrations of A T P and inorganic phosphate did not fall significantly during subsequent 90-minute incubation (Table I I ) . The leakage of total creatine and proteins is about 10 times more rapid during the first half hour than afterward. The observation that the ratios of 9F. D. Carlson, D. H. H a r d y , a n d D. R. Wilkie, J. (1967).
Physiol. (London) 189, 209
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TABLE II EFFECT OF INCUBATION AT 37 ° ON VARIOUS METABOLITES IN RAT DIAPHRAGMS WITHOUT RIBS a
Content of diaphragm (umoles/g)
ATP Phosphorylereatine Creatine Inorganic phosphate Tyrosine Ninhydrin-reaeting substances (leueine equivalents)
Initial
30 rain incubation
120 min incubation
4.1 9.3 9.0 7.4 0.17 26
3.2 9.8 4.7 6.8 0.13 18
3.0 7.7 2.8 7.8 0.13 14
Content of medium Total ereatine (uInoles/g wet weight) Protein (ing/mg dry weight)
0 0
4.4 0. 034
5.8 0. 044
, Quarter diaphragms from two or more animals were grouped for each experimental condition; means of two experiments are given. Methods of freezing the tissues, extraction, and analysis have been published elsewhere JR. Fulks, J. B. Li, and A. L. Goldberg, J. Biol. Chem. in press (1974); M. J. Kushmerick, R. E. Larson, and R. E. Davies, Proc. Roy. Soc. Ser. B 174, 293 (1969)]. A T P : t o t a l creatine and phosphocreatine:total creatine remained constant throughout the incubation period suggests t h a t the predominant number of cells maintain their energy reserves. In addition, intraeellular concentrations of several amino acids (e.g., phenylalanine and tyrosine) remain constant during incubation. Therefore, for studies of the diaphragm without ribs, in which measurements of the medium are to be made, a 30-minute preincubation of the tissue is recommended. Subsequently, the muscle is transferred to a second flask containing fresh medium, which is then closed and reequilibrated with the desired gas mixtures (see helow). Following this preincubarton, the rates of glucose oxidation, amino acid oxidation, protein synthesis, protein degradation, and nucleic acid synthesis are all linear for at least 2 hours. While m e m b r a n e potentials appear generally reduced (ranging between - - 4 0 and - - 5 0 mV), vigorous contractions can still be demonstrated for at least 4 hours. The actual physiological limitations of the diaphragms without ribs are unclear, since few studies have compared its properties with those of the intact preparation or the muscle in vivo. D i a p h r a g m s incut)ated without ribs synthesize proteins and actively transport amino acids and
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cations at normal or enhanced rates. It is, thus, incorrect to conclude that this preparation is generally leaky to small molecules or unstable as a consequence of injury to the fibers. The diaphragm without ribs also can respond to hormones, such as insulin, although the sensitivity of this preparation to endocrine factors may well be reduced. Nonspecific injury to muscle by itself has been shown to mimic certain actions of insulin2 ° Since other mammalian muscles can be studied without extraneous tissues attached and without cutting any fibers, comparative studies with such muscles may be performed to eliminate the possible effects of iniury.
Other Muscle Preparations Mammalian skeletal muscles have been reported to differ in important ways in their contractile and metabolic properties, apparently as a result of the differing types of muscle fibers of which they are composed. For example, in prolonged fasting or in response to large doses of glucocorticoids, the extensor digitorum longus and plantaris undergo marked atrophy, while the soleus shows little or no wasting. 11 Conclusions from studies of only the diaphragm are not necessarily valid for other muscles; for example, one unique property of the diaphragm is that upon denervation it undergoes a transient hypertrophy before the usual process of denervation atrophy. Similarly the levator anus muscle is uniquely dependent on the supply of androgens22 It, therefore, is important for many types of investigations to be able to compare several muscles of different physiological character. The isolated soleus, ~3,14 extensor digitorum longus, ~5 and gracilis anticus TM have all been used with success for metabolic or mechanical investigations. In addition, the rat peroneus longus, ~7 rat epitrochlaris, TM and mouse plantaris (unpublished) have been studied, and such preparations possibly merit further investigation. The anatomical locations of these muscles are found in Greene's atlas of rat anatomy29 Unfortunately the io W. D. Peckham and E. Knobil, Biochim. Biophys. Acta 63, 207 (1962). ~ A. L. Goldberg, in "Muscle Biology" vol. 1, pp. 84-118. Dekker, New York, 1972. ~2A. Arvill, S. Adolfsson, and K. Ahr6n, this volume [11]. ~3I. H. Chaudry and M. K. Gould, Biochim. Biophys. Acta 177, 527 (1969). ~A. L. Goldberg, C. M. Jablecki, and J. B. Li, Ann. N.Y. Acad. Sci. 228, 190 (1974). ~'~V. M. Pain and K. L. Manchester, Biochcm. J. 118, 209 (1970). ~"A. S. Bahler, J. T. Fales, and K. L. Zierler, J. Gen. Physiol. 51, 369 (1968). ~7K. L. Zierler, Amer. J. Physiol. 190, 201 (1957). ~*A. J. Garber, I. E. Karl, and D. M. Kipnis, Amer. J. Clin. Invest., 65th Meeting, p. 31a (1973). ~gE. C. Greene, "Anatomy of the Rat," The American Philosophical Society, Philadelphia, 1935; reprinted by Hafner, New York, 1963.
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substrate requirements and viability of these preparations have not been studied in any detail with the exception of the extensor digitorum longus, whose properties have been carefully investigated by Pain and Manchester. ~ The soleus and extensor digitorum longus (EDL) muscles are especially useful for comparative studies, since they are of relatively similar size and shape but contain substantially different proportions of fiber types (Fig. 1). Both are cylindrical fusiform structures, whose wet weights in a 50-60 g rat range between 20 and 30 rag. Since these muscles are significantly thicker than the diaphragm, use of rats larger than 60 g is not recommended. 1 In fact, upon incubation, EDL muscles exceeding 30 mg have been reported to show decreased ATP concentrations, poorer contractility, and increased extracellular fluid, unlike smaller muscles. 1~ The problems of diffusion of precursors into such thick muscles may also explain cer[ain published reports that the EDL contains multiple amino acid pools. .'° In composition the red soleus is one of the most uniform muscles of the body; approximately 80-90% of its fibers are of the slow twitch variety. Because these biochemical characteristics are adapted for continuous activity, the soleus may be a useful model for the behavior of cardiac muscle. On the other hand, the extensor digitorum longus is a pale muscle containing primarily fast twitch fibers and very few slow ones. Both muscles can be easily dissected without damage to the fibers and with tendons intact at each end of the muscle. The soleus lies beneath the larger gastrocnemius and plantaris on the dorsal side of the lower limb. Together with the gastrocnemius, the soleus inserts into the Achilles tendon. The soleus can be induced to hypertrophy by cutting the contributions of the synergistic gastrocnemius to this tendon. 11 In addition, the soleus can be easily denervated by sectioning the sciatic nerve in the thigh. The EDL lies on the ventrolateral aspects of the lower leg beneath the tibialis anterior. It is innervated by the common peroneal nerve, which can be easily .sectioned on the lateral aspect of the knee; compensatory hypertrophy of the EDL has been induced by tenotomy of the anterior tibialis. 21 The gracilis anticus, located on the ventromedial aspect of the thigh, has also been used for studies of muscle mechanics. The graeilis is thinner than the soleus or EDL, but its outer surface is covered by thick fascia, which may in vitro constitute a barrier for diffusion of nutrients. In addition, this muscle is more difficult to dissect and its tendons are shorter than those of the soleus and EDL. In fact, in order to avoid cutting any 2oj. B. Li, R. Fulks, and A. L. Goldberg, J. Biol. Chem. 248, 7272 (1973). 21E. Mackova and P. Hnik, Physiol. Bohemoslov. 21, 9 (1972).
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SKF~LETAL AND SMOOTH MUSCLE
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muscle fibers it is advisable to remove portions of the pubis where the muscle originates, and the femur, where it inserts. Conditions for Incubating Skeletal Muscles
These muscles have generally been incubated in Krebs-Ringer bicarbonate buffer 22 equilibrated with 95% ()2:5% C0_o and contain 119 mM NaC1, 5 mM KC1, 3 mM CaCl_~, 1 mM MgS04, 1 mM KH2P04, and 25 mM NaHC03 at pH 7.4. 25 Krebs-Ringer phosphate buffer equilibrated with 100% 02 has also been widely used. Chloramphenicol (0.3 ~g/ml) or other antibiotics are recommended for incubations lasting several hours, especially those employing labeled precursors. Addition of glucose (10 mM) to the incubation medium is advisable for incubations of several hours, although the diaphragm can oxidize its glycogen and triglyceride reserves for such periods in the absence of exogenous substrates. The addition of substrates is especially important for contracting muscle, whose metabolic requirements increase manyfold, and whose ability to contract may otherwise decrease rapidly. Various types of vessels appear suitable for these incubations; we have found 25-ml Erlenmeyer flasks quite convenient. For incubation of hemidiaphragms without ribs, 3 ml of medium should provide a large excess of nutrients. Obviously for the preparation with ribs, larger volumes of medium may be required. Although smaller flasks may allow use of less medium, under such conditions release of materials (e.g., amino acids or potassium) from the incubated muscles can markedly affect the composition of the medium. It is useful if the flasks are fitted with resealable rubber serum stoppers through which a hypodermic needle can be inserted to introduce materials. Through such a needle, the air trapped in the flask prior to incubation can be flushed out of the flask with either 02 or 95% 02:5% C02, while a second hypodermic needle provides an exit for the gas. The 20 ml of gas contained within such flasks contains 1 mmole of oxygen, which is far greater than that required by the diaphragm which, at rest, consumes 0.5 ~mole of 02 per gram per minute at 37 ° . Even though they are viable for 3-6 hours under these conditions, as evidenced by normal membrane potentials, ability to contract and to synthesize macromolecules, the diaphragm and other muscle preparations undergo net protein breakdown under these conditions. 23 In other words, protein degradation occurs several times more rapidly than protein syn~ W. W. Umbreit, R. H. Burris, and J. F. Stauffer, " M a n o m e t r i c Techniques," 4th ed. p. 131. Burgess, Minneapolis, Minnesota, 1964. 23 R. Fulks, J. B. Li, and A. L. Goldberg, J. Biol. Chem. (1974). I n press.
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thesis, and therefore these preparations leak amino acids into the medium. Addition of amino acids at the concentrations found in serum promotes protein synthesis, inhibits protein degradation, and helps maintain intracellular amino acid pools at normal levels. ~3 When amino acids are added at 5 times normal concentrations, the muscle approaches normal protein balance. Leucine, isoleueine, and valine appear to be the critical amino acids affecting protein balance in muscle, possibly because this tissue rapidly degrades these compounds, ~4 and they thus become rate limiting. Addition of glucose (10 raM) and insulin (0.1 unit/ml) also promote protein synthesis and inhibit degradation in these incubated tissues. Other metabolizable substrates, such as fatty acids or ketone bodies, have not yet been found to influence viability of the diaphragm. Pain and Manchester '~ have reported that albumin (0.13%) decreased potassium loss and promoted contractility of the EDL. Albumin may specifically interact with the muscle or may simply bind noxious agents, such as heavy metals, in the incubation medium. 15 The effects of other proteins on muscle viability have not been studied. One additional factor promoting muscle stability in vitro is the degree of tension (i.e., passive stretch) applied to the muscle. 1~,1~ Most investigators have incubated the diaphragm without any load, and such slack muscle tends to swell by taking up water. Passive tension of the diaphragm or extensor digitorum longus can retard this effect. 15 In addition, passive stretch of the diaphragm or soleus retards the net protein catabolism, characteristic of the incubated tissues54 Various groups have shown that stretch significantly enhances the long-term viability of incubated muscles from frog and rat. 1
SKELETAL AND SMOOTH MUSCLE
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gastrocnemius muscle were found to be well maintained during a 180minute perfusion. An example of the hormone responsiveness of the hemicorpus preparation is illustrated by the data shown in Fig. 4. In these experiments the effects of time of perfusion and insulin on the rate of protein synthesis in psoas muscle were studied. The rate of protein synthesis was linear during the first hour of perfusion, but declined sharply during the second and third hours (left panel, Fig. 4). The decline in the rate of protein synthesis was associated with increased numbers of ribosomal subunits and decreased numbers of polysomes (center and right panels, Fig. 4). The number of polysomes and ribosomal subunits is influenced by rates of initiation and elongation of peptide chains. Increased numbers of subunits and decreased numbers of polysomes result when initiation is retarded relative to elongation. 1~ These findings indicated that the decline in the rate of protein synthesis was due to a block in peptide-chain initiation which had developed during perfusion, perhaps as a result of depletion of hormones and substrates. Addition of insulin prevented development of the block in peptide-chain initiation as evidenced by maintenance of the initial rate of protein synthesis and in vivo levels of ribosomal aggregation. Other experiments indicated that glucose and fatty acid metabolism of the hemicorpus were also responsive to the in vitro addition of insulin. 14 The maintenance of the tissues of the hemicorpus in a good physiological state for perfusion periods up to 3 hours and the responsiveness of metabolic pathways to in vitro addition of hormone indicate the suitability of the preparation for studies of the metabolism of skeletal muscle. la H. E. Morgan, L. S. Jefferson, E. B. Wolpert, and D. E. Rannels, J. Biol. Chem. 246, 2163 (1971). 1~L. S. Jefferson, J. O. Koehler, and H. E. Morgan, Proc. Nat. Acad. Sci. U~S. 69, 816 (1972).
[10] I n V i t r o P r e p a r a t i o n s o f t h e D i a p h r a g m and Other Skeletal Muscles By ALFaED L. GOLDBERG,SUSAN B. MARTEL, and MARTIN J. KUSHMERICK Isolated mammalian muscles incubated in vitro offer numerous advantages for controlled investigations of muscle metabolism and neuromuscular physiology and pharmacology. This article will review certain prepa-
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r a t i o n s of skeletal muscle t h a t h a v e p r o v e d to be useful for biochemical a n d physiological studies in vitro. T h e m a j o r difficulty in m a i n t a i n i n g tissues in v i t r o has been e n s u r i n g t h a t a d e q u a t e a m o u n t s of n u t r i e n t s reach the i n d i v i d u a l cells b y diffusion in the absence of a c i r c u l a t o r y system. As a result, studies with i n c u b a t e d muscles have been restricted to especially t h i n muscles, o b t a i n e d from s m a l l rodents, t T h e v i a b i l i t y of such muscles in v i t r o g e n e r a l l y exceeds t h a t of slices from other m a m m a l i a n tissues, such as liver, k i d n e y , spleen, or brain. P e r f u s i o n of a specific muscle or whole h i n d l i m b s has also been used with success b y several laboratories2; however, the t e c h n i q u e s i n v o l v e d in isolation a n d i n c u b a t i o n of muscles in vitro are far simpler t h a n those r e q u i r e d for tissue perfusion. I n this discussion, special e m p h a s i s will be placed on the r a t diap h r a g m which has been the most f r e q u e n t l y s t u d i e d muscle p r e p a r a t i o n since it was first i n t r o d u c e d b y M e y e r h o f a n d H i m w i c h 3 n e a r l y fifty y e a r s ago. T h e i n c u b a t e d d i a p h r a g m was p o p u l a r i z e d p r i m a r i l y by the work of G c m m i l P a n d K i p n i s a n d Cori, 5 who d e m o n s t r a t e d t h a t it was a c o n v e n i e n t p r e p a r a t i o n for studies of h o r m o n a l control of c a r b o h y d r a t e m e t a b o l i s m a n d t r a n s p o r t . T h e m a j o r a d v a n t a g e s of the d i a p h r a g m are 'As a result of diffusion and metabolism, cells within an incubated muscle may be exposed to concentrations of nutrients different from those found in the incubation medium (A. V. Hill, "Trails and Trials in Physiology," p. 208, Williams & Wilkins, Baltimore, Maryland 1965). This difference will depend on the geometry of the tissue, the distance of a specific cell from the nearest surface, and the rate of utilization or release of substances from the cells. Thus in a flat tissue which consumps O2 at a rate A (measured in units of cm30~ STP/cm 3 tissue per minute), the distance X from the surface at which the O.~ concentration falls to zero is given by X = ( 2 K Y / A ) '/2, where K is Krogh's diffusion constant and Y is the O~ tension in the incubation medium measured as the partial pressure fraction. In muscle, K for O: is 1.66 × 10-~ cm~ per minute at 37°. For the rat diaphragm, basal O~ consumption rate at 37 ° is 0.5/zmole per gram per minute, and thus the critical depth into the muscle at which O~ tension falls to zero is 0.54 mm for incubations in 100% O~. For cylindrical muscles the appropriate formula is R = 2 ( K Y / A ) 1/0". Assuming similar rates of oxygen consumption for such muscles, the critical radius for in vitro incubations is about 0.77 mm. Since the Q10 for oxygen consumption is about 2.5 and for Krogh's constant K is about 1.1, it is clear that thicker muscles should be used only at temperatures below 37 ° to assure adequate oxygenation of all fibers; thus at 20 °, the critical distances are approximately twice that at 37 ° . 2L. S. Jefferson, this volume [91; J. T. Stull and S. E. Mayer, this volume [8]. 2, N. B. Ruderman, C. R. S. Houghton, and R. Hems, Biochem. J. 124, 639 (1971). ~O. Meyerhof and I-I. E. Himwieh, Pfluegers Arch. Gesamte Physiol. 205, 415 (1924). C. L. Gemmill, Bull. Johns Hopkins Hosp. 68, 329 (1941). D. M. Kipnis and C. F. Cori, J. Biol. Chem. 224, 681 (1957).
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SKELETAL AND SMOOTH MUSCLE
I A
B
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't c
FIG. 1. Drawings of rat diaphragm (A), extensor digitorum longus (B), and soleus (C), after L. T. Potter [J. Physiol. (London) 206, 145 (1970)] and R. A. Close [Physiol. Rev. 52, 129 (1972)]. Darkened areas indicate tendon. For mature rats (200 g) the calibration line adjacent to each muscle indicates 1 cm; in younger rats routinely used in our labs (50-70 g) the calibration is approximately 0.5 cm. The diaphragm is drawn as viewed from the thoracic cavity with the dorsal segment on top. Lines are drawn radially to indicate the orientation of the muscle fibers from the central tendon. The ribs have been removed. Note the more diffuse arrangement of nerve terminals (dots) in the right hemidiaphragm. In drawings of the extensor digitorum longus (B) and soleus (C) the right member of the pair represents a lateral view of the muscle; on the left are anteroposterior views. The distal tendons are uppermost in each case. Note that the soleus is thicker than the extensor digitorum longus. its rapid dissection and excellent viability in vitro. In addition, since the diaphragm can be readily dissected in combination with a considerable length of phrenic nerve, this preparation has been used widely by neurophysiologists and neurochemists. Various other preparations of mammalian skeletal muscle have been introduced. One major motivation for identifying other muscles that are convenient for study in vitro is that mammalian muscles are composed of at least three distinguishable types of fibers which differ in their physiological, histochemical, and ultrastructural properties2 Different muscles can v a r y in the relative proportions of the three fiber types and therefore can differ appreciably in their metabolic, endocrinological, and pharmacological responses. The Rat Diaphragm
Anatomy. In rats and mice, the diaphragm is an unusually thin sheet of muscle fibers that in young animals is even translucent. The muscle is composed of three distinct regions (Fig. 1) : two larger lateral portions, which are generally referred to as "hemidiaphragms," and a smaller dor6 R. A. Close, Physiol. Rev. 52, 129 (1972).
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sal segment adjacent to the spinal cord through which pass the esophagus, aorta, and vena eava. These subdivisions can be readily separated without damaging the fibers by cutting along the Y-shaped central tendon that delineates these three muscular segments. Within each hemidiaphragm, the muscle fibers run in a radial direction from the rib cage toward the central tendon. The neuromuscular junctions and the intradiaphragmatic portions of the phrenie nerve are localized in a well-defined thin band in the center of the muscle fibers that can be recognized with the naked eye (Fig. 1). In the diaphragm, red fibers predominate, but white and intermediate fibers each compose 20% of the total. Most investigators have discarded the smaller triangular segment of muscle and compared the responses of the contralateral hemidiaphragms using one as the control. It is also possible to cut the hemidiaphragm parallel to the muscle fibers so as to produce several segments of tissue from the same diaphragm. For most metabolic investigations in this laboratory, we routinely bisect the hemidiaphragm and compare the four "quarter diaphragms" under different experimental conditions. Thus far, the behavior of the "quarter diaphragm" has proved indistinguishable from that of the hemidiaphragm. Comparisons between segments from the same muscle offer obvious statistical advantages in reducing biologieal variability. Such internally controlled data are more difficult to obtain with other muscle preparations and impossible in perfused muscles. However, there are certain anatomical differences between the right and left hemidiaphragms. For example, the left hemidiaphragm with nerve attached has been used in most electrophysiological and biochemical studies of the neuromuscular junctions, because, on the left side, the region containing the nerve endings and neuromuscular junctions is more sharply delineated than on the right (Fig. 1). ~ In addition, the left phrenie nerve is simpler to dissect (or to section surgically) in the thorax than the right nerve. It thus seems advisable to cheek whether the behaviors of the contralateral hemidiaphragms are identical, at the outset of any investigation. Dissection o] the Diaphragm. In order to avoid problems of diffusion of nutrients into the incubated muscle, 1 it appears advisable to use rats weighing less than 100 g for most experiments. The best preparations of diaphragms from adult rats (280-320 g) lose most of their ATP and hexose phosphate intermediates upon incubationS; these findings contrast with those obtained with tissues from young rats (see below). For rectabolic studies, we routinely use 60-80 g rats (3-4 weeks of age), in which the wet weight of the hemidiaphragms ranges between 40 and 50 rag. L. T. Potter, J. Physiol. (London) 206, 145 (1970). K. A. Rookledge, Biochem. J. 125, 93 (1971).
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Such animals are rapidly growing, which complicates the planning of experiments. Obviously such rats are not appropriate for certain investigations (e.g., experiments on aging), and mouse diaphragm appears more appropriate for studies requiring adult tissues. For rapid removal of the diaphragm from small rats or mice, cervical dislocation by pressure with a rod behind the neck has proved to be a simple and rapid method for sacrificing the animal. Many labs have successfully employed decapitation or stunning by a blow to the head. The latter methods are not advised when diaphragm and phrenic nerve are to be removed together, since they can injure the nerve or cause hemorrhage and blood clotting within the thoracic cavity. For such dissections, anesthesia with ether or barbiturates appears preferable (e.g., 40-50 mg of pentobarbital per kilogram of rat weight with supplementary 10 mg doses as necessary). The diaphragm can be quickly excised along with a narrow ring of ribs to which the muscle is attached. After cutting through the skin on the ventral surface, the pectoral and abdominal muscles are trimmed from the rib cage. The lower edge of the diaphragm is next freed by cutting the several ligamentous attachments of the liver. The spinal column, esophagus, vena cava, and aorta are then severed below the diaphragm. The pleural cavity is entered by cutting through the sternum slightly above the xiphoid process, and the incision is extended on both sides to the spinal column. Finally the pericardial membranes and spinal column are severed above the diaphragm. The entire diaphragm with the supporting ribs and intercostal muscles is then transferred to a petri dish containing oxygenated buffer at room temperature. With practice, the time required from sacrifice to transfer to the petri dish can be shortened to less than 1 minute. The actual period of ischemia of the muscle is probably much shorter since the circulation remains intact until the aorta and vena cava are severed. The preparation in the petri dish is trimmed by removing the xiphoid process, spine, and dorsal segment of diaphragm. The central tendon is then bisected to yield two intact muscle masses. If necessary, the intercostal muscles and ribs are further trimmed to minimize the amount of tissue extraneous to the diaphragm. Since these associated tissues can weigh 10-50 times the hemidiaphragm, they may affect the composition of the incubation medium either by leaking or utilizing substances. The Diaphragm with and without Ribs
Unfortunately the diaphragm lacks a clearly defined tendon at the costal margins, and thus the investigator is forced either to incubate the
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SKELETAL
MUSCLE
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87
muscle with a portion of the ribs attached or to risk damage to the diaphragm by attempting to trim away the attached remnants of the rib cage. Both preparations have been used successfully for different purposes. For most studies it is advisable to incubate the hemidiaphragm with some ribs attached. As first shown by Kipnis and Cori, 5 such preparations more closely resemble the diaphragm in vivo and are more stable than hemidiaphragm or quarter diaphragm without ribs. The preparation with ribs is recommended for metabolic studies in which the products to be measured remain within the diaphragm, such as transport of substances into the intracellular space or incorporation of precursors into muscle protein, glycogen, fat, or nucleic acids. After incubation the diaphragm must then be cut away from the ribs, blotted, and weighed prior to chemical measurements. Incubation with ribs, however, cannot be used for studies in which the experimenter monitors loss of substances from the medium (such as measurements of 02 consumption) or evaluates metabolic products that might be released by the diaphragm into the surrounding media (such as measurements of transport out of the muscle, or investigation of CO~, lactate, and amino acid production). For such experiments, the diaphragm must be cut away from its supporting structures prior to incubation, since the greater mass of muscle and bone attached will leak materials into the medium and thus contribute to the values measured. Trimming of ribs must be performed with fine scissors as close to the costal border as possible so as to minimize damage to diaphragm and yet avoid the inclusion of intercostal muscles in the incubation. Gentle blotting and weighing of the tissue is advisable prior to incubation because such preparations often swell during the course of incubation. By a variety of criteria, the preparation with ribs more closely approximates the diaphragm within the organism than that with ribs removed, probably because the latter preparation contains appreciable numbers of injured fibers. (Kipnis and CorP have even termed this latter preparation the "cut diaphragm" to distinguish it from the "intact diaphragm" preparation with ribs.) For example, even after 90 minutes of incubation in oxygenated buffer, quarter diaphragms with ribs attached contain amounts of ATP, inorganic phosphate, and phosphorylcreatine similar to muscles removed immediately from the rat (Table I). In such diaphragms, the extracellular space, 5 the permeability to pentoses, 5 and total intracellular amino acids (estimated in ninhydrin-reactive material or fluoresceamine-reactive material) also did not fall significantly even after incubation for 120 minutes. In addition, the preparations with ribs can maintain nearly normal resting potentials for 4 hours, are capable
88
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S K E L E T A L AND SMOOTH MUSCLE
TABLE I COMPOSITION OF I~¢[ETABOLITES IN RAT DIAPHRAGM ~NCUBATED WITH AND WITHOUT RIBS a
Experimental conditions With ribs Initial 90-minuteincubation at 37° Without ribs Initial 90-minuteincubation at 37°
Creatine
Phosphorylcreatine
Total creatine
9.1 _+ 1 9.0_+ 0.2
12.5 + 1.2 10.8 + 0.5
21.6 + 0.5 4.3__ 0.5 19.8_+ 0.5 4.3 +_ 0.4
5.7 __ 1.l 6.7 _+ 0.2
8.9 __ 0.5 5.6_+ 0.5
12.2 ± 0.3 9.0_+ 0.9
21.3 + 0.4 4.0 +_ 0.5 14.7 _+ 1.3 3.5_+ 0.5
6.1 _+ 0.9 6.9 _+ 0.7
ATP
Inorganic phosphate
a Each value is the mean _+ SE of four determinations, each of which involved quarter diaphragms from three animals. Values are expressed as micromoles per gram of blotted wet weight. After freezing of the tissues in isopentane at - 150°C, the metabolites were extracted and analyzed by standard methods [M. J. Kushmerick, R. E. Larson, and R. E. Davies, Proc. Roy. Soc. Ser. B 174, 293 (1969)]. of contraction for up to 12 hours, and incorporate labeled amino acids into protein at constant rates for 4 hours. I n contrast, in diaphragms without ribs attached, the levels of creatine and phosphorylcreatine fall during incubation for 90 minutes. The total ereatine content (i.e., creatine and phosphorylcreatine combined) can be a useful measure of functional muscle mass, 9 and m a y even be a more reliable measure than wet weight, especially if some swelling or damage occurs. This fall in total creatine wouId suggest that some fibers in the muscle were damaged upon removal of the ribs and leaked metabolites into the medium. In support of this conclusion, creatine and phosphorylcreatine were recovered in the medium in amounts equal to those lost from the muscle (Table I I ) . Glucose, insulin, and amino acids at 5 times plasma concentrations did not prevent this leakage of total creatine. Furthermore, the preparation leaks several percent of its protein into medium. Although the diaphragm m a y leak intracellular components during the first half hour after the ribs are trimmed away, this preparation subsequently appears stable by several criteria. After the initial decrease, the concentrations of A T P and inorganic phosphate did not fall significantly during subsequent 90-minute incubation (Table I I ) . The leakage of total creatine and proteins is about 10 times more rapid during the first half hour than afterward. The observation that the ratios of 9F. D. Carlson, D. H. H a r d y , a n d D. R. Wilkie, J. (1967).
Physiol. (London) 189, 209
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OTHER
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TABLE II EFFECT OF INCUBATION AT 37 ° ON VARIOUS METABOLITES IN RAT DIAPHRAGMS WITHOUT RIBS a
Content of diaphragm (umoles/g)
ATP Phosphorylereatine Creatine Inorganic phosphate Tyrosine Ninhydrin-reaeting substances (leueine equivalents)
Initial
30 rain incubation
120 min incubation
4.1 9.3 9.0 7.4 0.17 26
3.2 9.8 4.7 6.8 0.13 18
3.0 7.7 2.8 7.8 0.13 14
Content of medium Total ereatine (uInoles/g wet weight) Protein (ing/mg dry weight)
0 0
4.4 0. 034
5.8 0. 044
, Quarter diaphragms from two or more animals were grouped for each experimental condition; means of two experiments are given. Methods of freezing the tissues, extraction, and analysis have been published elsewhere JR. Fulks, J. B. Li, and A. L. Goldberg, J. Biol. Chem. in press (1974); M. J. Kushmerick, R. E. Larson, and R. E. Davies, Proc. Roy. Soc. Ser. B 174, 293 (1969)]. A T P : t o t a l creatine and phosphocreatine:total creatine remained constant throughout the incubation period suggests t h a t the predominant number of cells maintain their energy reserves. In addition, intraeellular concentrations of several amino acids (e.g., phenylalanine and tyrosine) remain constant during incubation. Therefore, for studies of the diaphragm without ribs, in which measurements of the medium are to be made, a 30-minute preincubation of the tissue is recommended. Subsequently, the muscle is transferred to a second flask containing fresh medium, which is then closed and reequilibrated with the desired gas mixtures (see helow). Following this preincubarton, the rates of glucose oxidation, amino acid oxidation, protein synthesis, protein degradation, and nucleic acid synthesis are all linear for at least 2 hours. While m e m b r a n e potentials appear generally reduced (ranging between - - 4 0 and - - 5 0 mV), vigorous contractions can still be demonstrated for at least 4 hours. The actual physiological limitations of the diaphragms without ribs are unclear, since few studies have compared its properties with those of the intact preparation or the muscle in vivo. D i a p h r a g m s incut)ated without ribs synthesize proteins and actively transport amino acids and
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SKELETAL AND SMOOTH MUSCLE
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cations at normal or enhanced rates. It is, thus, incorrect to conclude that this preparation is generally leaky to small molecules or unstable as a consequence of injury to the fibers. The diaphragm without ribs also can respond to hormones, such as insulin, although the sensitivity of this preparation to endocrine factors may well be reduced. Nonspecific injury to muscle by itself has been shown to mimic certain actions of insulin2 ° Since other mammalian muscles can be studied without extraneous tissues attached and without cutting any fibers, comparative studies with such muscles may be performed to eliminate the possible effects of iniury.
Other Muscle Preparations Mammalian skeletal muscles have been reported to differ in important ways in their contractile and metabolic properties, apparently as a result of the differing types of muscle fibers of which they are composed. For example, in prolonged fasting or in response to large doses of glucocorticoids, the extensor digitorum longus and plantaris undergo marked atrophy, while the soleus shows little or no wasting. 11 Conclusions from studies of only the diaphragm are not necessarily valid for other muscles; for example, one unique property of the diaphragm is that upon denervation it undergoes a transient hypertrophy before the usual process of denervation atrophy. Similarly the levator anus muscle is uniquely dependent on the supply of androgens22 It, therefore, is important for many types of investigations to be able to compare several muscles of different physiological character. The isolated soleus, ~3,14 extensor digitorum longus, ~5 and gracilis anticus TM have all been used with success for metabolic or mechanical investigations. In addition, the rat peroneus longus, ~7 rat epitrochlaris, TM and mouse plantaris (unpublished) have been studied, and such preparations possibly merit further investigation. The anatomical locations of these muscles are found in Greene's atlas of rat anatomy29 Unfortunately the io W. D. Peckham and E. Knobil, Biochim. Biophys. Acta 63, 207 (1962). ~ A. L. Goldberg, in "Muscle Biology" vol. 1, pp. 84-118. Dekker, New York, 1972. ~2A. Arvill, S. Adolfsson, and K. Ahr6n, this volume [11]. ~3I. H. Chaudry and M. K. Gould, Biochim. Biophys. Acta 177, 527 (1969). ~A. L. Goldberg, C. M. Jablecki, and J. B. Li, Ann. N.Y. Acad. Sci. 228, 190 (1974). ~'~V. M. Pain and K. L. Manchester, Biochcm. J. 118, 209 (1970). ~"A. S. Bahler, J. T. Fales, and K. L. Zierler, J. Gen. Physiol. 51, 369 (1968). ~7K. L. Zierler, Amer. J. Physiol. 190, 201 (1957). ~*A. J. Garber, I. E. Karl, and D. M. Kipnis, Amer. J. Clin. Invest., 65th Meeting, p. 31a (1973). ~gE. C. Greene, "Anatomy of the Rat," The American Philosophical Society, Philadelphia, 1935; reprinted by Hafner, New York, 1963.
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substrate requirements and viability of these preparations have not been studied in any detail with the exception of the extensor digitorum longus, whose properties have been carefully investigated by Pain and Manchester. ~ The soleus and extensor digitorum longus (EDL) muscles are especially useful for comparative studies, since they are of relatively similar size and shape but contain substantially different proportions of fiber types (Fig. 1). Both are cylindrical fusiform structures, whose wet weights in a 50-60 g rat range between 20 and 30 rag. Since these muscles are significantly thicker than the diaphragm, use of rats larger than 60 g is not recommended. 1 In fact, upon incubation, EDL muscles exceeding 30 mg have been reported to show decreased ATP concentrations, poorer contractility, and increased extracellular fluid, unlike smaller muscles. 1~ The problems of diffusion of precursors into such thick muscles may also explain cer[ain published reports that the EDL contains multiple amino acid pools. .'° In composition the red soleus is one of the most uniform muscles of the body; approximately 80-90% of its fibers are of the slow twitch variety. Because these biochemical characteristics are adapted for continuous activity, the soleus may be a useful model for the behavior of cardiac muscle. On the other hand, the extensor digitorum longus is a pale muscle containing primarily fast twitch fibers and very few slow ones. Both muscles can be easily dissected without damage to the fibers and with tendons intact at each end of the muscle. The soleus lies beneath the larger gastrocnemius and plantaris on the dorsal side of the lower limb. Together with the gastrocnemius, the soleus inserts into the Achilles tendon. The soleus can be induced to hypertrophy by cutting the contributions of the synergistic gastrocnemius to this tendon. 11 In addition, the soleus can be easily denervated by sectioning the sciatic nerve in the thigh. The EDL lies on the ventrolateral aspects of the lower leg beneath the tibialis anterior. It is innervated by the common peroneal nerve, which can be easily .sectioned on the lateral aspect of the knee; compensatory hypertrophy of the EDL has been induced by tenotomy of the anterior tibialis. 21 The gracilis anticus, located on the ventromedial aspect of the thigh, has also been used for studies of muscle mechanics. The graeilis is thinner than the soleus or EDL, but its outer surface is covered by thick fascia, which may in vitro constitute a barrier for diffusion of nutrients. In addition, this muscle is more difficult to dissect and its tendons are shorter than those of the soleus and EDL. In fact, in order to avoid cutting any 2oj. B. Li, R. Fulks, and A. L. Goldberg, J. Biol. Chem. 248, 7272 (1973). 21E. Mackova and P. Hnik, Physiol. Bohemoslov. 21, 9 (1972).
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SKF~LETAL AND SMOOTH MUSCLE
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muscle fibers it is advisable to remove portions of the pubis where the muscle originates, and the femur, where it inserts. Conditions for Incubating Skeletal Muscles
These muscles have generally been incubated in Krebs-Ringer bicarbonate buffer 22 equilibrated with 95% ()2:5% C0_o and contain 119 mM NaC1, 5 mM KC1, 3 mM CaCl_~, 1 mM MgS04, 1 mM KH2P04, and 25 mM NaHC03 at pH 7.4. 25 Krebs-Ringer phosphate buffer equilibrated with 100% 02 has also been widely used. Chloramphenicol (0.3 ~g/ml) or other antibiotics are recommended for incubations lasting several hours, especially those employing labeled precursors. Addition of glucose (10 mM) to the incubation medium is advisable for incubations of several hours, although the diaphragm can oxidize its glycogen and triglyceride reserves for such periods in the absence of exogenous substrates. The addition of substrates is especially important for contracting muscle, whose metabolic requirements increase manyfold, and whose ability to contract may otherwise decrease rapidly. Various types of vessels appear suitable for these incubations; we have found 25-ml Erlenmeyer flasks quite convenient. For incubation of hemidiaphragms without ribs, 3 ml of medium should provide a large excess of nutrients. Obviously for the preparation with ribs, larger volumes of medium may be required. Although smaller flasks may allow use of less medium, under such conditions release of materials (e.g., amino acids or potassium) from the incubated muscles can markedly affect the composition of the medium. It is useful if the flasks are fitted with resealable rubber serum stoppers through which a hypodermic needle can be inserted to introduce materials. Through such a needle, the air trapped in the flask prior to incubation can be flushed out of the flask with either 02 or 95% 02:5% C02, while a second hypodermic needle provides an exit for the gas. The 20 ml of gas contained within such flasks contains 1 mmole of oxygen, which is far greater than that required by the diaphragm which, at rest, consumes 0.5 ~mole of 02 per gram per minute at 37 ° . Even though they are viable for 3-6 hours under these conditions, as evidenced by normal membrane potentials, ability to contract and to synthesize macromolecules, the diaphragm and other muscle preparations undergo net protein breakdown under these conditions. 23 In other words, protein degradation occurs several times more rapidly than protein syn~ W. W. Umbreit, R. H. Burris, and J. F. Stauffer, " M a n o m e t r i c Techniques," 4th ed. p. 131. Burgess, Minneapolis, Minnesota, 1964. 23 R. Fulks, J. B. Li, and A. L. Goldberg, J. Biol. Chem. (1974). I n press.
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thesis, and therefore these preparations leak amino acids into the medium. Addition of amino acids at the concentrations found in serum promotes protein synthesis, inhibits protein degradation, and helps maintain intracellular amino acid pools at normal levels. ~3 When amino acids are added at 5 times normal concentrations, the muscle approaches normal protein balance. Leucine, isoleueine, and valine appear to be the critical amino acids affecting protein balance in muscle, possibly because this tissue rapidly degrades these compounds, ~4 and they thus become rate limiting. Addition of glucose (10 raM) and insulin (0.1 unit/ml) also promote protein synthesis and inhibit degradation in these incubated tissues. Other metabolizable substrates, such as fatty acids or ketone bodies, have not yet been found to influence viability of the diaphragm. Pain and Manchester '~ have reported that albumin (0.13%) decreased potassium loss and promoted contractility of the EDL. Albumin may specifically interact with the muscle or may simply bind noxious agents, such as heavy metals, in the incubation medium. 15 The effects of other proteins on muscle viability have not been studied. One additional factor promoting muscle stability in vitro is the degree of tension (i.e., passive stretch) applied to the muscle. 1~,1~ Most investigators have incubated the diaphragm without any load, and such slack muscle tends to swell by taking up water. Passive tension of the diaphragm or extensor digitorum longus can retard this effect. 15 In addition, passive stretch of the diaphragm or soleus retards the net protein catabolism, characteristic of the incubated tissues54 Various groups have shown that stretch significantly enhances the long-term viability of incubated muscles from frog and rat. 1
