Regional differences in myosin heavy chain isoforms and enzyme activities of the rat diaphragm TAKAO SUGIURA, SHUNSUKE MORITA, AK10 MORIMOTO, AND NAOTOSHI MURAKAMI Laboratory of Biomechanics and Physiology, College of General Education, Yamaguchi University, Yamaguchi 753; and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan SUGIURA, TAKAO, SHUNSUKE MORITA, AKIO MORIMOTO, AND NAOTOSHI MURAKAMI. Regional differences in myosin heavy chain isoforms and enzyme activities of the rat diaphragm. J. Appl. Physiol. 73(2): 506-509, 1992.-Myosin heavy chain isoforms and enzyme activities were compared between the costal and crural regions of the rat diaphragm. The percentage of heavy chain (HC) IIb in the crural region of the diaphragm was significantly (P < 0.05) higher than that in the costal region (mean 7.3 vs. 3.0%), and the percentage of HCI was significantly lower in the crural than in the costal diaphragm (22.7 vs. 27.9%). The distributions of HCIIa and HCIId were relatively homogeneous in both regions. Succinate dehydrogenase activity in the costal diaphragm was 21% greater (P < 0.01) than in the crural diaphragm. In contrast, there was no significant difference in the activity of phosphofructokinase in the crural and costal diaphragms. These results demonstrate that a difference in myosin heavy chain isoforms and oxidative capacity exists between the costal and crural regions of the rat diaphragm. succinate tivity
dehydrogenase
activity;
phosphofructokinase
ac-
THE DIAPHRAGM, which has a crucial role in respiratory
action, basically consists of two muscles, the costal and the crural. Each muscle is composed of three types of fibers, which are classified by histochemical staining (10, 12,13,1&l& 19,24). Although there have been a number of studies on the regional difference in fiber-type composition of the diaphragm, disagreements exist in the published results (12, 15, 18, 19, 24). The discrepancies may be explained by differences in the fiber-type classification schemes and/or animal species used. It is generally accepted that the histochemical fiber types, classified by the difference in the pH sensitivity of the myofibrillar adenosinetriphosphatase (ATPase) reaction, are correlated with the presence of different myosin heavy chain (MHC) isoforms (2,521). Recently, using a gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Bar and Pette (1) demonstrated that the diaphragm contains an abundance of a newly discovered fast MHC isoform, tentatively designated as heavy chain (HC)IId. To our knowledge, no information has been available concerning regional differences in MHC isoforms, including HCIId, of the rat diaphragm. Moreover it seems important to understand whether a difference in metabolic properties, in addition to MHC expression, exists between the costal and crural dia506
phragms. The mean succinate dehydrogenase (SDH) activities in type IIb fibers are lower than that in either type I or IIa fibers, although considerable overlap in the distributions of SDH activities has been shown in type IIa and IIb fibers in the diaphragm (13, 23). More recently, Larsson et al. (14) showed that the average SDH activities in the 11x fiber containing MHC-2X, which is considered to be identical to HCIId, were intermediate between those of the type IIa and IIb fibers of the rat tibialis anterior muscle. If these relationships apply to the diaphragm and MHC composition differs between the costal and crural diaphragms, then it would be expected that a regional metabolic difference exists in the diaphragm. In this regard, only a few studies have been performed on the enzyme activities of the costal and crural diaphragms (17,25). However, it remains controversial whether there are regional differences in enzyme activity between the costal and crural diaphragms. Sieck et al. (25) found no regional differences in the SDH activity of the cat diaphragm, whereas Powers et al. (17) demonstrated regional variability in the rat diaphragm. Hence, the purpose of this investigation was to characterize the rat diaphragm with regard to MHC isoform distribution and metabolic enzyme activities. METHODS Animals. These experiments were approved by the University Committee for Use of Animals in Research and followed the guidelines established by the American Physiological Society. Twenty male Wistar rats, 10 wk old, were purchased from Kyudo (Saga, Japan). Mean body weight and diaphragm weight were 306.7 t 4.8 (SE) g and 891.0 t 18.9 mg, respectively. Sample preparation. Animals were killed with ether, and the entire diaphragm was rapidly removed and placed in ice-cold saline. The excised muscle was quickly dissected free of fat and tendon. Although a regional anatomic model for the rat diaphragm has been proposed by Metzger et al. (15), some investigators found no regional differences in fiber-type composition or enzyme activities within the rat costal diaphragm (15,17). In the present study, therefore, we examined the right crural diaphragm and the entire right costal diaphragm. The divided segments were quickly frozen in isopentane precooled in liquid nitrogen and were stored at -80°C until analyzed. Enzyme assays. The activities of two enzymes, SDH
0161-7567192 $2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/jappl at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.
MYOSIN
1 Ila IId& Ilb I/-
2
HEAVY
CHAIN
AND ENZYME
ACTIVITY
OF DIAPHRAGM
507
1. Myosin heavy chain isoforms in costal and crural regions of rat diaphragm
3
TABLE
Ila 1/IId -Ilb -1
1. Electrophoretograms of myosin heavy chain (MHC) isoforms from costal and crural regions of rat diaphragm. Lanes: 1, mixture of plantaris and diaphragm; 2, crural diaphragm; 3, costal diaphragm. Only area of MHC region is shown. FIG.
(E.C. 1.3.99.1) and phosphofructokinase (PFK, E.C. 2.7.1.56) were used to determine oxidative and glycolytic potentials, respectively. Tissue samples used to determine SDH activity were homogenized in ice-cold 33.3 mM phosphate buffer (1:20 wt/vol, pH 7.4). The tissue used for PFK activity was homogenized in ice-cold homogenization medium containing 150 mM KCl, 50 mM KHCO,, and 6 mM EDTA (1:20 wt/vol). SDH activity was determined using the spectrophotometric method described by Cooperstein et al. (4) with cytochrome c as the terminal hydrogen acceptor. SDH was assayed using 20 ~1 of tissue homogenate. The reaction medium for SDH consisted of 40.5 mM phosphate buffer (pH 7.4), 16.6 mM sodium succinate, 1 mM NaCN, 16.6 PM cytochrome c, 0.4 mM AlCl,, and 0.4 mM CaCl,. The reduction of cytochrome c was followed for 3 min at a wavelength of 550 nm. PFK activity was assayed spectrophotometrically based on the rate of endogenous NADH oxidation (22). The PFK reaction medium consisted of 50 mM triethanolamine (pH 7.6), 5 mM EDTA, 10 mM MgCl,, 2.8 mM ATP, 2.8 mM fructose 6-phosphate, 0.3 mM NADH, 1 U aldolase, and a 17.1-U a-glycerophosphate dehydrogenase-18.3-U triosephosphate isomerase mixture. The reaction was started by the addition 20 ~1 of homogenate and followed for 3 min at a wavelength of 340 nm. All enzyme assays were performed in triplicate at 25°C and the results were expressed in micromoles per gram of noncollagenous protein (NCP) per minute. One milliliter of the homogenate was used for the determination of NCP content (27). Protein concentration was determined by a protein assay kit (Bio-Rad) with bovine y-globulin as the standard. The intra-assay coefficients of variation for SDH, PFK, and NCP were 3.6,4.0, and 2.0%, respectively. MHC analysis. Analysis of MHC isoforms was carried out electrophoretically as described by Bar and Pette (1) and Sugiura and Murakami (28). Briefly, SDS-PAGE was performed on slab gel (7 cm X 9 cm X 1 mm) by use of a 5-8% (wt/vol) polyacrylamide-30-40% (vol/vol) glycerol gradient-separating gel and a 3.5% (wt/vol) polyacrylamide stacking gel containing 35% (vol/vol) glycerol. The residual homogenate used to determine SDH activity was centrifuged, and the sediment was rehomogenized (1:40, wt/vol) in an SDS sample buffer containing a final concentration of 30% (vol/vol) glycerol, 5% (vol/vol) fl-mercaptoethanol, 2.3% (wt/vol) SDS, 62.5 mM tris(hydroxymethyl)aminomethane-HCl (pH 6.8), and 0.05% (wt/vol) bromphenol blue. The homogenate
Myosin
Heavy
Chain Isoforms
Region
I
IIa
IId
IIh
Costa1 Crural
27.9*1.5 22.7+-1.4*
31.1kl.l 3O.Ok1.2
37.9k1.5 4O.Bl.6
3.OkO.8 7.3+1.7*
Values are means rir SE in percent; n = 20. * Statistically (P < 0.05) from costal diaphragm.
different
was incubated for 10 min at -60°C and further diluted 1:25 with the same buffer. A lo-p1 sample of protein was then applied to the gel. Electrophoresis was first run at 50 V/plate until the tracking dye completely entered the separating gel and then at 150 V/plate for - 18 h at 8°C. Gels were stained in a solution containing 0.1% (wt/vol) Coomassie Brilliant Blue, 50% (vol/vol) methanol, and 10% (vol/vol) acetic acid and destained by diffusion in a solution of 5% (vol/vol) methanol and 7% (vol/vol) acetic acid. The percent distribution of MHC isoforms was calculated using a thin-layer chromatography scanner (model CS-930, Shimadzu, Kyoto, Japan) equipped with a laser source attachment (LSA-9000, Shimadzu). Analysis of the MHC isoforms was performed in triplicate, and the intra-assay coefficients of variation for each MHC isoform were as follows: HCI, 4.7%; HCIIa, 2.6%; HCIId, 3.2%: HCIIb, 10.8%. St&istical ‘analysis. Statistical significance was determined by Student’s unpaired two-tailed t test performed on the means. Differences were considered statistically significant if P < 0.05. RESULTS
Figure 1 and Table 1 show electrophoretograms and percentages of MHC isoforms in the costal and crural regions of rat diaphragm, respectively. The MHC isoforms of adult skeletal muscle are separated into four types, HCIIa, HCIId, HCIIb, and HCI, in order of increasing electrophoretic mobility on 5-8% gradient SDSPAG (32). All four MHC isoforms were observed in the costal and the crural diaphragm; however, the percentages of MHC isoforms differed between the costal and the crural diaphragm; the costal diaphragm contained significantly (P < 0.05) more HCI and less HCIIb than the crural diaphragm. In contrast, there was no significant difference in the relative distribution of HCIIa and HCIId between the costal and the crural diaphragm. SDH and PFK activities in the costal and the crural regions of the rat diaphragm are shown in Table 2. SDH activity was significantly (P < 0.01) higher in the costal than in the crural region of the diaphragm. In addition, 2. PFK and SDH activities regions of rat diaphragm
TABLE
in costal and crural
Region
PFK Activity
SDH Activity
Costa1 Crural
94.9k3.4 99.6zi4.6
27.4k1.5 22.6fO.S*
Values are means * SE in pmol . g noncollagenous protein-‘. mini; Statistically different (I’ < 0.01) from costal diaphragm.
n = 20. *
Downloaded from www.physiology.org/journal/jappl at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.
508
MYOSIN
HEAVY
CHAIN
AND ENZYME
although the difference was not significant, PFK activity tended to be higher in the crural than in the costal region of the diaphragm. DISCUSSION
ACTIVITY
OF DIAPHRAGM
differences in the SDH activity of the cat diaphragm. The discrepancy may be explained by differences in animal species, as suggested by Powers et al., and/or in the method used to determine SDH activity. Using microphotometric techniques, Green et al. (13) demonstrated that the mean SDH activities of type I fibers are significantly higher than those of type IIb fibers in the rat costal diaphragm. From the studies by Green et al. and Metzger et al. (l5), a regional difference in SDH activity might be anticipated on the basis of the relative distribution of MHC isoforms shown in this study. In contrast, it is generally accepted that the glycolytic enzyme activity is higher in fast-twitch fibers of locomotor muscles than in slow-twitch fibers (20). If this is true of the rat diaphragm, it is to be expected that the PFK activity is higher in the crural than in the costal diaphragm. In this study, however, the PFK activity tended to be higher in the crural than in the costal diaphragm, but the difference was not significant. The reason for this is unknown. We observed regional differences in HCI, HCIIb, and SDH activity between the costal and the crural diaphragm, although these differences were very small. There is controversy regarding whether the two regions of the diaphragm have different recruitment patterns during respiratory and nonrespiratory functions (6-9,11, 16, 24-26). Whether these small regional differences in heavy chain isoforms and enzyme activities contribute to regional differences in diaphragmatic function can be elucidated only with further study.
The results demonstrate the presence of regional differences in the percent distributions of MHC isoforms in the rat diaphragm, although the differences were small. The percentages in the distribution of HCI and HCIIb isoforms were different in the costal and crural regions of the diaphragm of the rat, and there were no regional differences in the relative distribution of HCIId and HCIIa. Metzger et al. (15) reported that the rat crural diaphragm contains significantly more type IIb fibers and fewer type I fibers than the costal region. It is generally accepted that MHC isoforms determine the histochemical fiber types proposed by Brook and Kaiser, with HCI, HCIIa, and HCIIb being contained in type I, IIa, and IIb fibers, respectively (2, 5, 21). Furthermore, using the criteria of histochemical fiber typing according to Brooke and Kaiser, Termin et al. (29) recently reported that fibers containing the HCIId isoform were almost indistinguishable from, and could be classified histochemically as, type IIb fibers. If this is assumed to be true, the total percentage of HCIIb and HCIId obtained in the present study is significantly higher in the crural than in the costal region (47.4 vs. 41.4%, P < 0.05). Consequently the findings with respect to MHC isoforms in this study support the existence of regional differences in fiber-type We thank Prof. T. Yoshioka (St. Marianna University School of distribution reported by Metzger et al. Medicine) for valuable comments and Dr. N. C. Long (Yamaguchi UniMetzger et al. (15) also reported that although the his- versity School of Medicine) for review of the English manuscript. tochemical fiber-type distribution was different in the Address for reprint requests: T. Sugiura, Laboratory of Biomechancostal and in the crural diaphragm, no significant differits and Physiology, College of General Education, Yamaguchi Univerences were detected in maximum contraction speed sity, Yamaguchi 753, Japan. (V,,,>. One possible explanation for their findings is Received 25 February 1991; accepted in final form 2 March 1992. that, of the fibers classified histochemically as type IIb by Metzger et al., few fibers contain HCIIb, because most of REFERENCES the type IIb fibers in the rat diaphragm contain HCIId 1. BAR, A., AND D. PETTE. Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett. 235: 153-155, 1988. (29). More recently, Bottinelli et al. (3) reported that the 2. BILLETER, R., C. W. HEIZMANN, H. HOWALD, AND E. JENNY. AnalVmax of type 11x fibers containing MHC-2X, considered ysis of myosin light and heavy chain types in single human skeletal to be identical to HCIId, was lower than that of type IIb muscle fibers. Eur. J. Biochem. 116: 389-395, 1981. fibers and that type IIa and 11x fibers had similar mean 3. BOTTINELLI, R., S. SCHIAFFINO, AND C. REGGIANI. Force-velocity relations and myosin heavy chain isoform composition of skinned Vmax values. They also showed that type II fibers had fibres from rat skeletal muscle. J. Physiol. Land. 437: 655-672, overlapping Vmax ranges. As shown in Table 1, the sum of 1991. HCIIa and HCIId is ~70% of the whole population and S. J., A. LAZAROW, AND N. J. KURFESS. A micro4. COOPERSTEIN, is similar in both diaphragm regions. Judging from these spectrophotometric method for the determination of succinic dehyfindings, it might be inferred that the regional differdrogenase. J. Biol. Chem. 186: 129-139, 1950. 5. DANIELI-BETTO, D., E. ZERBATO, AND R. BETTO. Type 1,2A, and ences in the proportion of type I and IIb fibers in the 2B myosin heavy chain electrophoretic analysis of rat muscle costal and crural diaphragms is insufficient to produce fibers. Biochem. Biophys. Res. Commun. 138: 981-987,1986. the regional difference in Vmax. S. G. KELSEN, G. S. SUPINSKI, AND 6. DARIAN, G. B., A. F. DIMARCO, The present study also showed higher oxidative capacS. B. GOTTFRIED. Effects of progressive hypoxia on parasternal, costal, and crural diaphragm activation. J. Appl. Physiol. 66: 2579ity in the costal than in the crural region but similar 2584,1989. glycolytic enzyme activity in both regions. This finding is 7. DE TROYER, A., M. SAMPSON, S. SIGRIST, AND P. T. MACKLEM. quite consistent with the results of a recent study by The diaphragm: two muscles. Science Wash. DC 213: 237-238, Powers et al. (l7), who reported that SDH activity is 1981. higher in the costal than in the crural rat diaphragm, but 8. DE TROYER, A., M. SAMPSON, S. SIGRIST, AND P. T. MACKLEM. Action of costal and crural parts of the diaphragm on the rib cage in they found no differences in lactate dehydrogenase activdog. J. AppZ. PhysioZ. 53: 30-39, 1982. ity between these two regions. As far as SDH activity is 9. EASTON, P. A., J.-W. FITTING, AND A. E. GRASSINO. Costa1 and concerned, however, our results and those of Powers et crural diaphragm in early inspiration: free breathing and occlusion. al. (17) conflict with the findings of Sieck et al. (25), who, J. AppZ. Physiol. 63: 1622-1628, 1987. using microphotometric techniaues, found no regional 10. EDWARDS, R. H. T., AND J. A. FAULKNER. Structure and function Downloaded from www.physiology.org/journal/jappl at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.
MYOSIN
11. 12. 13.
14.
HEAVY
CHAIN
AND ENZYME
of the respiratory muscles. In: The Thorax, edited by C. Roussos and P. T. Macklem. New York: Dekker, 1985, pt. A, p. 297-326. (Lung Biol. Health Dis. Ser.) FOURNIER, M., AND G. C. SIECK. Somatotopy in the segmental innervation of the cat diaphragm. J. Appl. Physiol. 64: 291-298,1988. GEORGE, J., AND A. SUSHEELA. A histophysiological study of the rat diaphragm. Biol. Bull. Woods Hole 121: 471-480, 1961. GREEN, H. J., H. REICHMANN, AND D. PETTE. Inter- and intra-speties comparison of fiber type distribution and succinate dehydrogenase activity in type I, IIA and IIB fibers of mammalian diaphragms. Histochemistry 81: 67-73, 1984. LARSSON, L., L. EDSTROM, B. LINDEGREN, L. GORZA, AND S. SCHIAFFINO. MHC composition and enzyme-histochemical and physiological properties of a novel fast-twitch motor unit type. Am.
J. Physiol. 15. METZGER,
261 (Cell Physiol. 30): C93-ClOl, J. M., K. B. SCHEIDT, AND R.
and physiological
characteristics
1991.
H. FITTS. Histochemical of the rat diaphragm. J. AppZ.
Physiol. 58: 1085-1091, 1985. 16. MONGES, H., J. SALDUCCI, AND
B. NAUDY. Dissociation between the electrical activity of the diaphragmatic dome and crura muscular fibers during esophageal distention, vomiting and eructation. An electromyographic study in the dog. J. Physiol. Paris 74: 541-
554, 1978. 17. POWERS, FORSTER,
S. K., J. LAWLER, D. CRISWELL, H. SILVERMAN, H. V. S. GRINTON, AND D. HARKINS. Regional metabolic differences in the rat diaphragm. J. AppZ. Physiol. 69: 648-650, 1990. 18. REID, W. D., J. M. HARDS, B. R. WIGGS, E. N. WOOD, P. V. WRIGHT, AND R. L. PARDY. Proportions and sizes of muscle fiber types in the hamster diaphragm. MuscZe Nerve 12: 108-118, 1989. 19. RILEY, D. A., AND A. J. BERGER. A regional histochemical and electromyographic analysis of the cat respiratory diaphragm. Exp. Neural.
66: 636-649,
1979.
ACTIVITY
OF DIAPHRAGM
509
20. SALTIN, B., AND P. D. GOLLNICK. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. SkeZetaZ Muscle. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 10, p. 555-631. G., R. BETTO, D. DANIELI-BETTO, AND M. ZEVIANI. 21. SALVIATI, Myofibrillar-protein isoforms and sarcoplasmic-reticulum Ca2+transport activity of single human muscle fibers. Biochem. J. 224: 215-225,
1983.
22. SHONK, C. E., AND G. E. BOXER. I. Methods for the determination
Enzyme patterns in human tissue. of glycolytic enzymes. Cancer Res.
24: 709-724,1964. 2% SIECK, G. C., AND
C. E. BLANCO. Postnatal changes in the distribution of succinate dehydrogenase activities among diaphragm muscle fibers. Pediatr. Res. 29: 586-593, 1991. 24. SIECK, G. C., R. R. ROY, P. POWELL, C. BLANCO, V. R. EDGERTON, AND R. M. HARPER. Muscle fiber type distribution and architecture of the cat diaphragm. J. AppZ. Physiol. 55: 1386-1392, 1983. 25. SIECK, G. C., R. D. SACKS, AND C. E. BLANCO. Absence of regional differences in the size and oxidative capacity of diaphragm muscle 1987. 26 fibers. J. AppZ. Physiol. 63: 1076-1082, SIECK, G. C., R. B. TRELEASE, AND R. M. HAPPER. Sleep influences on diaphragmatic motor unit discharge. Ecp. Neural. 85: 316-335, l
1984. 27. SUGITA,
H., Y. OKUMURA, AND K. AYAI. Application of a property of troponin to determination of tropomyosin content of a small piece of muscle. J. Biochem. 65: 971-972, 1969. 28 SUGIURA, T., AND N. MURAKAMI. Separation of myosin heavy chain isoforms in rat skeletal muscles by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biomed. Res. 11: 87-91, l
1990.
29. TERMIN, A., R. S. STARON, AND D. PETTE. Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Histochemistry
92: 453-457,1989.
Downloaded from www.physiology.org/journal/jappl at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.