Histochemical types and sizes of fibers in the rectus abdominis muscle of guinea pigAdaptive response to pregnancy.код для вставкиСкачать
THE ANATOMICAL RECORD 217:23-29 (1987) Histochemical Types and Sizes of Fibers in the Rectus Abdominis Muscle of Guinea Pig: Adaptive Response to Pregnancy G. LALATTA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI Institute of Veterinary Anatomy, University of Bologna, 40126 Bologna, Ztaly ABSTRACT Effects of pregnancy stimulation upon histochemically assessed myofibi-illar ATPase and muscle fiber diameters were analysed in the rectus abdominis (RA) muscle of guinea pig. Samples of the muscle were taken a t 30, 40, 50, 60, and 70 days of pregnancy and compared with samples of the same muscle taken from nonpregnant guinea pigs. Changes in muscle fiber proportions were noted through the course of pregnancy. Starting from 50 days of gestation a n increase in type I fibers and a decrease in type IIB fibers were noted. Increase in muscle fiber diameters was also observed in type I, IIA, and IIB fibers. In addition, the RA muscle of the male guinea pig was compared with that of the female guinea pig and showed more type IIA and less type IIB fibers and all the three fiber types were larger than those of the female. In recent years a number of studies of skeletal muscle have demonstrated that fiber type transformation is possible not only with cross-innervation and specific electrical stimulation (for references see Jolesz and Sreter, 1981; Mabuchi et al., 1982) but also with prolonged and intense endurance training. In animals (Kowalski et al., 1969; Barnard et al., 1970; Faulkner et al., 1972; Maxwell et al., 1973; Muller, 1974; Green et al., 1984) and man (for references see Howald, 1982) the effects of endurance training consisted in a n increase in aerobic metabolism and/or transformation of type IIB into type IIA fibers and also of type I1 into type I fibers. Pregnancy, with its increase in abdominal contents, may represent a physiological chronic stimulus imposed on the muscles of the abdominal wall which should result, in our opinion, in a fiber type transformation. Data on this subject are not available, with the exception of a study by Martin (1979), who has been unable to detect a significant change in rectus abdominis (RA) muscle fiber proportions through the course of pregnancy in the rat. However, in this animal pregnancy lasts only 21 days. In order to verify whether a longer gestation period could result in a muscle fiber transformation, we investigated the effect of pregnancy on RA muscle of the guinea pig, as in this laboratory animal the pregnancy lasts approximately 70 days, i.e., a time period which should be long enough to allow possible changes in muscle fibers. RA muscle was chosen since it is the most involved muscle in any stretch of the abdominal wall. A preliminary report has been given elsewhere (Bortolami et al., 1980). The fiber type patterns in the RA muscle of male and female guinea pig was also compared to investigate possible sex differences. 0 1987 ALAN R. LISS, INC. MATERIALS AND METHODS Animals and Muscle Samples Adult nulliparous female and male albino guinea pigs of the same age and same weight were used. Females were placed in a cage with male guinea pigs for a period of 3 days and the middle of the 3 days was counted as day 0 of pregnancy. The animals, killed with a n overdose of Nembutal, were 1) three females at 30, 40, 50, 60, and 70 days of pregnancy, 2) nine nonpregnant control females, and 3) nine males. The control females and the males were killed at time points corresponding to days 0,40, and 70 of gestation of the pregnant animals. Both the RA muscles were dissected from each subject with their full abdominal length from the cartilago xifoidea to the cranial level of the pubis and a mediolatera1 extent from the linea alba to the superficial epigastric arteries. The muscles appeared clearly segmented by six or seven spaced tendinous intersections. The muscle samples were removed from the central portion of each segment in order to avoid the areas near the tendinous intersections and rapidly frozen by immersion in isopentane cooled with liquid nitrogen. Serial cross sections were cut at 10 K r n on a cryostat microtome at - 20°C. Enzyme Histochemistry and Fiber Typing Serial sections were assayed for myofibrillar adenosine triphosphatase (ATPase) activity following alkaline (pH 10.4) and acid (pH 4.6) preincubation (Brooke and Kaiser, 1970). Sections were also incubated for succinic acid dehydrogenase (SDH) (Nachlas et al., 1957) and Received February 25, 1986; accepted August 4, 1986. 24 G. LALATTA COSTERBOSA. A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI menadione-linked a-glycerophosphate dehydrogenase (a-GPDH) (Hess and Pearse, 1961).According to the terminology of Brooke and Kaiser (1970), four muscle fiber types were considered (types I, IIA, IIB, IIC). Figure 1 illustrates the results of the staining procedures used in this study. The type I fibers were dark after acid preincubation (Fig. 1A)but light after alkaline preincubation (Fig. 1B); type IIA fibers showed the reverse pattern; type IIB fibers were dark at pH 10.4 but intermediate at pH 4.6. Fibers classified as type IIC remained stable throughout the entire pH range used. The type IIAB fibers were also observed and grouped with the type IIB fibers (see Staron et al., 1984). Oxidative activity was high in type I, IIA, and IIC fibers (Fig. lC), while glycolytic activity was low only in type I fibers (Fig. 1D). In order to determine the percentage of each fiber type, different areas of each RA muscle segment had been randomly selected. The proportion of each muscle fiber type was calculated as a percentage of the total number of fibers in each segment analysed. A total of about 4,000 fibers were considered for each subject. The same areas utilized for the fiber type counting were then photographed and x 100 magnification pictures were used to obtain the mean fiber diameters for 60 fibers of each type in each subject. The diameter measured was the maximum diameter across the lesser aspect of the muscle fiber (Brooke, 1970). This diameter was chosen in preference to fiber area because it is not subject to enlargement by oblique sectioning. The data obtained from individual segments of RA in each animal were pooled; this was possible since no significant differences occurred between the different segments in each animal. After having statistically tested the homogeneity of the data within each experimental group by the one-way analysis of variance, the results from the different groups were analysed and compared with the controls by using Student’s t-test (significance was accepted at the P < .05, P < .01,P < .001 levels). RESULTS Muscle Fiber Types Fiber typing was based on the ATPase reaction after acid preincubation (Fig. 2). As can be seen in Table 1, RA muscle of both sexes was composed predominantly of type 11fibers, type IIB being prevalent; type IIC fibers were always very few. The males, compared with the females, showed more type IIA and less type IIB fibers while no significant difference in percentage of type I fibers was observed. In control animals killed at different moments corresponding to either 0 or 40 or 70 days of pregnancy, no differences were observed in the percentages of RA muscle fiber types. Changes in muscle fiber proportions were noted through the course of pregnancy (Table 1).In fact, type I fibers underwent first (30 days) a decrease followed by a slow but consistent progressive increase in number (Fig. 3C), which was statistically significant starting from 50 days of pregnancy. Type IIA fibers remained essentially the same in number and showed a significant decrease only at 60 days of pregnancy. The proportion of type IIl3 fibers increased up to 40 days of pregnancy and then underwent a progressive decrease which became statistically significant at 70 days (Fig. 4). Muscle Fiber Diameters In the RA muscle of both sexes the type IIB fibers showed the largest diameter, while type I fibers presented the smallest diameter (Table 2). Significant differences were observed between the male and female (Table 2): all the three fiber types were larger in male than in female (Fig. 3A,B). In control animals killed at different moments corresponding to either 0 or 40 or 70 days of pregnancy, no differences were observed in muscle fiber diameters. Changes in muscle fiber diameters were observed during pregnancy in each muscle fiber type (Figs. 2, 3C, 5). The type I, IIA, and IIB fiber diameters increased significantly starting from 50 days of pregnancy. At the end of pregnancy the type I fibers showed the greatest relative increase in diameter (30.80%), followed by type IIA (8.99%) and type IIB (6.65%) fibers (Table 2). DISCUSSION The guinea pig RA muscle of both sexes contains a high proportion of type I1 fibers which are also generally larger than type I fibers. The male shows significantly more type IIA and less type IIB fibers and all the three fiber types are significantly larger than those of the female. In this investigation, the purpose of using the pregnancy of the guinea pig was to determine the effect of a long-term stimulation, such as the effect of a 70-day pregnancy, on the RA muscle. Data were collected at 30, 40, 50, 60, and 70 days of pregnancy. At 30-40 days of pregnancy a transient slight decrease of type I fibers and conversely a n increase of type IIB fibers were observed. Starting from 50 days of pregnancy the pattern changed and a progressive increase in the proportions of type I and a progressive decrease of type IIB fibers occurred. Concerning the fiber diameters, all three fiber types increased significantly starting from 50 days of pregnancy and type I exhibited the greatest relative increase. It has been shown that fast muscles subjected to chronic electrical stimulation exhibit a remarkable increase in their capacity for aerobic metabolism (Peckham et al., 1973; Pette et al., 1973; Riley and Allin, 1973) and a n increase in number of slow fibers (Munsat et al., 1976; Rubinstein et al., 1978). Changes in the ultrastructure of myofibrils as a result of a changed impulse pattern have also been shown (Salmons et al., 1978; Heilman and Pette, 1979; Sjostrom et al., 1980). Recently, Green et al. (1984) have suggested that 15 weeks of a treadmill training program can provoke a n increase in type I with a concomitant decrease in type IIB fibers, which has been not only histochemically but also biochemically assessed. Taken collectively, the results of all these studies show that inyreased activity is capable of inducing true fiber type transformation. This remarkable plasticity of the skeletal muscle fiber has been histochemically demonstrated also in the present study. The RA muscle, in fact, during pregnancy shows first a n adaptation which involves mostly the type IIB fibers and successively, when the continuous stimulation produced by pregnancy exerts its influence on the abdominal wall in its stronger form, the muscle then acquires a histochemical pattern, with increased type I fibers, which appears to suit it to its new functional role, in which a weight-bearing activity becomes GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY Fig. 1. Serial cross sections from rectus abdominis (RA) muscle of guinea pig (control female) showing the staining patterns of type I, type IIA, type IIB, type IIAB, and type IIC fibers. A) ATPase, section preincubated at pH 4.6. B) ATPase, section preincubated at pH 10.4.C) Succinic acid dehydrogenase (SDH). D) Alpha-glycerophosphatedehydrogenase (a-GPDH).X 190. 25 26 G. LALATTA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI Fig. 2. RA muscle of guinea pig. A) Control female. B) Regnant (50 days) female. C) Regnant (60 days) female. D) Pregnant (70 days) female. A-D) ATPase, section preincubated at pH 4.6. In the pregnant females there is a n increase in diameter of all fiber types. A muscle spindle is identifiable in A (bottom right of the photograph). x 100. 27 GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY TABLE 1. Percentage distribution of fibers in rectus abdominis (RA) muscle of nonpregnant female, male, and pregnant female guinea pig' Sex F contr. M FP. FP. FP. FP. FP. Days of gestation Type 1 0 31.69 32.13 29.46* 30.10 33.94" 36.35* * * 38.64** * - 30 40 50 60 70 (%o) Type IIA Type IIB (%I (%I Type IIC (%I 21.10 23.71*** 21.40 19.50 22.09 18.83* 20.17 46.77 43.73*** 48.78* 50.17*** 43.56 44.18 40.78*** 0.44 0.43 0.36 0.23 0.41 0.64 0.41 'Abbreviations: F contr., nonpregnant control female; M, male; Fp., pregnant female. *Significant difference from control females (P < .05). ***Significant difference from control females ( P < ,001). TABLE 2. Mean fiber diameters of RA muscle of nonpregnant female, male, and pregnant female guinea pig' Sex F contr. M FP. FP. FP. FP. FP. Days of gestation 0 30 40 50 60 70 Type 1 M+SE (pm) 41.88 & 0.39 43.59 & 0.42* 42.63 0.65 40.86 i 0.65 53.89 + 0.61**'k 50.58 + 0.58**'k 54.78 i 0.61**'& Type IIA M+SE Type IIB M+SE (clm) Gm) 54.06 k 0.40 58.72 f 0.48* 54.04 k 0.79 54.28 f 0.65 61.19 k 0.80*** 58.31 + 0.69*** 58.92 + 0.71*** 57.90 & 0.42 69.09 + 0.51*** 55.54 + 0.88 58.81 f 0.72 65.56 + 0.78*** 60.36 + 0.66* 61.75 f 0.73*** lAbbreviations: F contr., nonpregnant control female; M,male; Fp., pregnant female. *Significant difference from control females ( P < . O W ***Significant difference from control females ( P < ,001). fundamental. The earlier transient increase in type IIB fibers observed at 30-40 days of pregnancy could be interpreted as a change regulated at least in part by the involvement of endocrine secretions, such as the sex hormones and thyroid hormones, since their effect on muscle fiber sizes and types has been clearly shown (see Kelly, 1983). Starting from 50 days of pregnancy, the stretch of RA muscle in combination with the functional overload due to the sizes and weight of the fetuses could prevail over any possible hormonal influence and result in a n increase of type I fibers. The increased proportion of type I fibers might result either from a transformation of type I1 into type I fibers or from the formation of new fibers. However, since ectopic nuclei, fissures, groups of fibers having a much reduced diameter, and all the aspects which have been generally reported to occur in the process leading to fiber division (Hall-Craggs, 1972) were never observed in our material, the latter hypothesis, in our opinion, is rather unlikely. The other possibility is that a fiber transformation could take place. According to this hypothesis, the increase in number of type I fibers could be gradually generated via IIC fibers by transformation from type IIA (Jansson et al. 1978; Pierobon-Bormioli et al., 1981) and a conversion of type IIB via IIAB to IIA fibers (Ingjer, 1979) could also occur. This hypothesis could be also supported by the immunological and biochemical studies, which demonstrated that both fast and slow myosin isozymes are present within single fibers during transformation provoked by long-term electrical stimulation (Pette and Schnez, 1977; Rubinstein et al., 1978), thus indicating that a transformation can take place within the preexisting set of fibers. Moreover, the RA muscle of the guinea pig at 50-70 days of pregnancy has significantly larger fibers of all three types than the same muscle of nonpregnant animals. Since it is well known that fiber diameter is related to the forces the fibers are required to develop (Saltin and Gollnick, 1983) and that the only resource available for increasing total muscular strength is to induce hypertrophy of the existing muscle fibers, the hypertrophy of RA muscle fibers noted in pregnant guinea pig should demonstrate that relatively high forces are continuously produced. These forces are necessary to maintenance of tone in the abdominal wall a s the RA muscle is stretched during pregnancy. Finally, the duration of the pregnancy stimulation is fundamental; in fact, while the 21-day pregnancy of the rat provoked no change on RA muscle fiber proportions and hypertrophy of only type I fibers CMartin,l979), our data suggest that the long-term pregnancy of guinea pig plays a significant role in RA muscle fibers, which react to changes in functional demand with both type transformation and hypertrophy. In fact, when pregnancyinduced stretch and work become remarkable the hypertrophy of all the fiber types takes place and the greater relative increase in diameter of the type I fibers shows a preferential recruitment of this fiber type. Concomitantly, a n increase in number of this fiber type also appears to be necessary and shows that the muscle becomes adapted for constant use, can contract and relax more slowly, and is more fatigue-resistant. 28 G. LALAYIA COSTERBOSA, A.M. BARAZZONI, M.L. LUCCHI, AND R. BORTOLAMI -1 ---DA ......DE 0 30 40 DAYS OF m w N o I 50 60 70 Fig. 4. Mean frequencies of RA muscle fiber types in the guinea pig during pregnancy. *Level of significance (P < .05).***Level of significance (P < .001). E 5 70 I 30 40 50 60 70 DAYS OF PREGNANCY Fig. 5. Mean diameters of RA muscle fiber types in the guinea pig during pregnancy. *Level of significance (P < .05). ***Level of significance (P < .001). Fig. 3. RA muscle of guinea pig x 100.A) Male guinea pig. B) Control female. C) Pregnant (70 days) female. A-C) ATPase, section preincubated at pH 10.4. Type I fibers stain light; note that they are larger and more numerous in the pregnant female. The male shows larger fibers compared with the control female. A muscle spindle is identifiable in C (top portion of the photograph). GUINEA PIG RECTUS ABDOMINIS MUSCLE IN PREGNANCY ACKNOWLEDGMENTS The authors wish to thank Prof. P. Monari of the Department of Statistical Sciences, University of Bologna, for statistical analysis of data. The authors also express their gratitude to Ms. M.L. Polsoni for typing the manuscript and to Mr. E. Ferrari for his technical assistance. This research was supported by grants from C.N.R. and M.P.I. of Italy. LITERATURE CITED Barnard, R.J., V.R. Edgerton, and J.B. Peter (1970) Effect of exercise on skeletal muscle. I. Biochemical and histochemical properties. J. Appl. Physiol., 28:762-766. Bortolami, R., V. De Pasquale, and G. Lalatta Costerbosa (1980)Effect of pregnancy on muscle fibers of the rectus abdominis muscle of the guinea pig. Boll. Soc. Ital. Biol. Sper., 56:1322-1325. Brooke, M.H. (1970) Some comments on neural influence on the two histochemical types of muscle fibre. In: The Physiology and Biochemistry of Muscle as a Food. E.J. Briskey, R.G. Cassens, and B.B. Marsh, eds. Madison, University of Wisconsin Press, Vol. 2, pp. 131-153. Brooke, M.H., and K.K. Kaiser (1970) Muscle fiber types: How many and what kind? Arch. Neurol., 23:369-379. Faulkner, J.A., L.C. Maxwell, and D.A. Lieberman (1972) Histochemical characteristics of muscle fibers from trained and detrained guinea pigs. Am. J. Physiol., 222:836-840. Green, H.J., G.A. Klug, H. Reichmann, U. Seedorf, W. Wiehrer, and D. Pette (1984) Exercise-induced fibre type transitions with regard to myosin, parvalbumin, and sarcoplasmic reticulum in muscles of the rat. Pfliigers Arch., 400t432-438. Hall-Craggs, E.C.B. (1972) The significance of longitudinal fibre division in skeletal muscle. J. Neurol. Sci., 15:27-33. Heilman, C., and D. Pette (1979) Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation. Eur. J. Biochem., 93:437-446. Hess, R., and A.G.E. Pearse (1961)Histochemical and homogenization studies of mitochondrial a-glycerophosphate dehydrogenase in the nervous system. Nature, 191:718-719. Howald, H. (1982) Training-induced morphological and functional changes in skeletal muscle. Int. J. Sports Med., 3:l-12. Ingjer, F. (1979) Effects of endurance training on muscle fibre ATP-ase activity, capillary supply and mitochondria1 content in man. J. Physiol. (Lond.),294:419-432. Jansson, E., B. Sjodin, and P. Tesch (1978) Changes in muscle fibre type distribution in man after physical training. A sign of fibre type transformation? Acta Physiol. Sand., 104t235-237. Jolesz, F., and F.A. Sreter (1981) Development, innervation, and activity-pattern induced changes in skeletal muscle. Annu. Rev. Physiol., 43:531-552. Kelly, A.M. (1983) Emergence of specialization in skeletal muscle. In: Handbook of Physiology. Skeletal Muscle. L.D. Peachey, ed. Ann. 29 Physiol. Soc., Bethesda, Maryland, Sect. 10, Chap. 19, pp. 507-537. Kowalski, K., E.E. Gordon, A. Martinez, and J. Adamek (1969) Changes in enzyme activities of various muscle fiber types in rat induced by different exercises. J. Histochem. Cytochem., 17:601-607. Mabuchi, K., D. SzveLko, K. Pinter, and F.A. Sreter (1982) Type IIB to IIA fiber transformation in intermittently stimulated rabbit muscles. Am. J. Physiol., 242:C373-C381. Martin, W.D. (1979) A study of the effect of pregnancy on muscle fibers of the rectus abdominis muscle of the rat. Anat. Rec., 195t455-462. Maxwell, L.C., J.A. Faulkner, and D.A. Lieberman (1973) Histochemical manifestations of age and endurance training in skeletal muscle fibers. Am. J. Physiol., 224t356-361. Miiller, W. (1974)Temporal progress of muscle adaptation to endurance training in hind limb muscles of young rats. Cell Tissue Res., f56:61-87. Munsat, T.L., D. McNeal, and R. Waters (1976) Effects of nerve stimulation on human muscle. Arch. Neurol., 33:608-617. Nachlas, M.M., K.C. Tsou, E. De Souza, C.S. Cheng, and A.M. Seligman (1957)Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem., 5,420-436. Peckham, P.H., J.T. Mortimer, and J.P. Van der Meulen (1973) Physiologic and metabolic changes in white muscle of cat following induced excercise. Brain Res., 50:424-429. Pette, D., and U. Schnez (1977) Coexistence of fast and slow type myosin light chains in single muscle fibres during transformation as induced by long term stimulation. FEBS Lett. 83:128-130. Pette, D., M.E. Smith, H.W. Staudte, and G.Vrbova (1973) Effects of long-term electrical stimulation on some contractile and metabolic characteristics of fast rabbit muscles. Pfliigers Arch., 338:257-272. Pierobon-Bormioli, S., S. Sartore, L. Dalla Libera, M. Vitadello, and S. Schiafho (1981) “Fast” isomyosins and fiber types in mammalian skeletal muscle. J. Histochem. Cytochem., 29:1179-1188. Riley, D.A., and E.F. Allin (1973)The effects of inactivity, programmed stimulation, and denervation on the histochemistry of skeletal muscle fiber types. Exp. Neurol., 40:391-413. Rubinstein, N., K. Mabuchi, F. Pepe, S. Salmons, J. Gergely, and F. Sreter (1978) Use of type-specific antimyosins to demonstrate the transformation of individual fibers in chronically stimulated rabbit fast muscles. J. Cell Biol., 79:252-261. Salmons, S., D.R. Gale, and F.A. Sreter (1978) Ultrastructural aspects of the transformation of muscle fibre type by long term stimulation: Changes in 2 discs and mitochondria. J. Anat., 127:17-31. Saltin, B., and P.D. GoIlnick (1983)Skeletal muscle adaptability: Significance for metabolism and performance. In: Handbook of Physiology. Skeletal Muscle. L.D. Peachey, ed. Am. Physiol. SOC., Bethesda, Maryland, Sect. 10, Chap. 19, pp. 555-631. Sjostrom, M., A.C. Edman, and S. Salmons (1980) Changes in M-Band structure accompanying the transformation of rat fast muscle by long-term stimulation. Muscle Nerve Abstr., 3:277. Staron, R.S., R.S. Hikida, F.C. Hagerman, G.A. Dudley, and T.F. Murray (1974) Human skeletal muscle fiber type adaptability to various workloads. J. Histochem. Cytochem., 32:146-152.