Acta Physiol Scand 1992, 144, 419423
Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat T. S U G I U R A , H. MATOBA*, H. MIYATA, Y. K A W A I and N. M U R A K A M I t Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, * Faculty of Integrated Arts and Sciences, Tokushima University, Tokushima and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
+
SUGIURA, T., MATOBA, H., MIYATA, H., KAWAI, Y. & MURAKAMI, N. 1992. Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiol Scand 144, 419423. Received 26 August 1991, accepted 3 December 1991. ISSN 0001-6772. Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, Faculty of Integrated Arts and Sciences, Tokushima University, Tokushima, and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan. Using gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis, myosin heavy chain (MHC) isoforms were studied in the extensor digitorum longus (EDI,) and the soleus muscles of male Wistar rats at different ages (5, 10, 20 weeks, 1 and 2 years). In the EDL muscle, four types ofMHC isoforms were observed in all age groups. There was an increase in the percentage of HCIId and a concomitant decrease in the percentage of HCIIb with increasing age. No significant difference was observed in the percentages of HCI and HCIIa isoforms in all the age groups. In contrast, the soleus muscle contained two MHC isoforms, HCI and HCIIa. There was an increase in the percentage of HCI and a concomitant decrease in the percentage of HCIIa with increasing age. These results suggest that age-related changes in the MHC isoforms in both the fast-twitch EDI, and the slow-twitch soleus muscles are one factor underlying the age-related decrease in the speed of muscle contraction. Key words : ageing, fast-twitch muscle, myosin heavy chain isoforms, rat, slow-twitch muscle.
I t is well known that the speed of muscle contraction decreases during the ageing process (Gutmann & Syrovy 1974, Larsson & Edstrom 1986, Edstrom & Larsson 1987, Larsson & Salviati 1989). A decrease in the proportion of the fast-twitch fibres is considered to be one possible mechanism underlying the age-related decrease in the speed of contraction (Kugelberg 1976, Eddinger et a/. 1985, Larsson & Edstrom 1986, Edstrom & Larsson 1987, Ishihara & Araki 1988). However, this is not a consistent phenomCorrespondence : Takao Sugiura, Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi 753, Japan.
enon of ageing in all muscles; it is generally reported that the slow-twitch soleus muscle shows an age-related decrease in the relative proportion of fast-twitch fibres (Kugelberg 1976, Eddinger et al. 1985), whereas the fast-twitch extensor digitorum longus (EDL) muscle does not show such alterations (Eddinger et a / . 1985, Larsson & Edstrom 1986, Ishihara & Araki
1988). T h e histochemical actomyosin myofibrillar adenosine triphosphatase (ATPase) reaction of muscle fibres is determined by the difference of myosin heavy chain (MHC) isoforms (Billeter et al. 1981, Danieli-Betto et al. 1986). Recently, a new fast MHC isoform was found in most fast-
419
420
7'.Siigiura
et al.
T a b l e 1. Body and muscle weights of animals at five postnatal periods Postnatal age
nod!
1st
(g)
EI)I. \\t (mg) Soleus u t ( m g ) \ alue5 are means
. irieek5
10 \seeks
20 necks
1 !ear
2 years
144.0k 2.; 66.8 1 .2 57.4+0.7
317.4k8.2 1 5 4 . 0 i 5.9
497.2 k 9.9 211.3kY.5 189.9 7.9
6.53.6k 11.5 279.2 i 1 .0 2 3 7 . i i 10.1
6.50.8k3.5..5 230.8 k 16.9 186.0f 18.0
*
140.9
*
0.9
+SE, N = 5
tv itch muscles using gradient sodium dodecyl sulphate-.polvacrylamide gel electrophoresis (SDS---P.qGE;Bar & Pette 1988). This isoform was tentativel!- designated as H C I I d , since it was detected prcdominantl!- in the adult rat diaphragm. Similarh, Schiafino rt al. (1989) reported the existence of a third MHC, designated as 2X-MHC by the use of monoclonal antibodies specific for MHC. T h e fact that these \lHC isoforms are identical has been suggested by inimunoblot analysis (LaFramboise rt (11. 1990). I t is also suggested that these isoforms ha\-c intcrmediate features between the HCIIb and H C I I a isoforms (Termin rt a / . 1989a & b, Larsaon et ul. 1991). This leads to the assumption that the relative concentration of H C I I d or 2XVHC increases as the speed of contraction decreases during the ageing process. C'nfortunately, to date no information is a\-ailable concerning this. Therefore, the present study was undertaken to clarify the changes with age of the MHC isoforms, including HCIId, in fastand slow-twitch muscles of the rat,
41.A 'YE R I -4 I, S .4 ND 11F: 'I' H 0 D S :Jnimals. These experiments followed the guidelines for thc care and use of animals in the field of ph! siological sciences (The Physiological Society of Japan). Five groups of five male IVistar rats, aged 5, 10, 70 weeks, 1 and 2 years, w r e used in this stud!(see Table 1). They were purchased from K y d o Co. I,td (Saga, Japan). The animals were killed bl- ether inhalation and weighed. After weighing, the soleus and EDI, muscles were r e m o d , and were quickly dissected frec of fat and tendon. The dissected muscles were weighed and frozen in isopentane precooled in liquid nitrogen. They were then stored at -80 "C: until the analysis of XIIIC isoforms was conducted. . y u w p [ ~prc'plrrcrfion. ~ Using a glass homogenizer, a portion of the muscle was homogenized in 10 \-ol of
ice-cold buffer containing 0.1 M KCI, 1 mM E D T A and 20 m \ i Tris-llaleate buffer (pH 7.0). T h e homogenate itas centrifugcd at 1000 g for 10 niin at 4 '(;, and the pellet a a s rehomogenized (1 :40, w/v) in an SDS sample buffer containing 30"" (v/v) glycerol, 5 O , , (v/\-) /I-mercaptoethanol, 2.3 "() (\v/v) SDS, 62.5 m u Tris-HC1 (pII 6.8) and 0.05", (w/v) bromophenol blue. The homogenate was incubated for 10 niin at about 60 "C, and further diluted 1 : 2.5 with the same bufler. Then a 7.5/ t l portion of the homogenate was electrophoresed. SDS-P.4GE. T h e analysis of M H C isoforms was electrnphorcticalll- carried out according to the method of Bar & Pette (1988) as modified by Sugiura & llurakami (1990). Briefly, SDS-P.4CiE was performed on a slab gel (7 x 9 cm x 1 mni) using a S4",,, ( w / v ) polya~rylarnide/3(WO",~ (v/v) gly-cerol gradient separating gel and a 3.j0,, (w/v) polyacrylamide stacking gel containing 35 "(,(v/v) glycerol. Electrophoresis w s first run at 50 V until the tracking dye cornpierel>- entered the separating gel, and then at 1.50 V for about 18 h at 8 "C. Gels were stained in a solution containing 0. 1 O 0 ( w / v ) Coomassie brilliant blue, 50c1(,(../I) methanol, and lo",, (v/v) acetic acid, and destained b!- diffusion in a solution of S",, ( v / v ) methanol and 7",, (\-/\-) acetic acid. T h e percentage distribution of MI I C isoforms was calculated b!- the use of a Shimadzu CS-930 TLC Scanner (Kyotn, Japan) equipped with a laser source attachment (ISA9000). S/o/rs/ic.s. Means and standard errors were calculated from indi\ idual values by standard procedures. A one-nay anall-sis of variance (ANOVA) was performed in order to evaluate the significance of ageassociated trends. Tukey's method was used for intergroup comparison. The 0.05 confidence level was chosen for statistical significance.
RESULTS Figure 1 and Table 2 show the electrophoretogram and the percentage of MHC isoforms in the EDI, and soleus muscles, respecti\ely. XIHC isoforms of the EDJ, muscle
Ageing rat myosin heazy chain
421
( P < 0.05) and the percentage of H C I I a was significantly lower (P < 0.05) compared with the 5-week-old soleus muscle. DISCUSSION Fig. 1. Electrophoretograms of myosin heavy chain (MHC) isoforms from the rat extensor digitorum longus (EDL) and soleus muscles. Lanes: 1, 5-weekold EDL; 2,2-year-oId EDL; 3, 5-week-old soleus; 4,
2-year-old soleus. Only the area of the MHC region is shown.
are separated into four types, HCIIa, H C I I d , H C I I b and HCI, in order of increasing electrophoretic mobility, on a 5-80/, gradient SDSPAGE (Termin et al. 1989a & b). There was no change in the percentage of HCI and HCII (IIb, I I d and IIa) isoforms in the EDL muscle from the 5-week to the 2-year age groups. However, there were small changes in the percentages of HCII isoforms with increasing age. T h e percentage of H C I I d was significantly higher at 1 and 2 years than at 5 weeks, whereas the percentage of H C I I b was significantly lower (P< 0.05) at 1 and 2 years than at 5 weeks. No significant difference was observed in the percentage of H C I I a between the age groups. I n contrast to the E D L muscle, the soleus muscle contained only two MHC isoforms, HCI and HCIIa, at 5 weeks of age, HCI being more prominent than HCIIa. Twenty weeks later, the percentage of HCI was significantly higher
Histochemical studies using rat skeletal muscles have shown that in the soleus muscle the percentage of type I fibres increases during development (Eddinger et al. 1985), whereas in the EDL muscle fibre type differentiation occurs by approximately 3 weeks postpartum (Kelly 1983), and the transformation of muscle fibres from type I1 to type I does not occur during subsequent development (Eddinger et al. 1985, Larsson & Edstrom 1986, Ishihara & Araki 1988). T h e histochemical myofibriliar ATPase reaction of a muscle fibre is determined by its myosin heavy chain composition (Billeter et al. 1981, Danieli-Betto et al. 1986). Recent reports (Termin et al. 1989a) have also shown that type I I D fibres containing H C I I d are almost indistinguishable from type I I B fibres containing HCIIb, using the criteria of Brooke & Kaiser (1970). I n this study, as shown in Table 2 , there was an increase in the percentage of HCI and a concomitant decrease in the percentage of HCIIa with increasing age in the soleus muscle. O n the other hand, in the E D L muscle, there were no changes in the sum of H C I I b and H C I I d from the 5-week to the 2-year age groups. Assuming that fibres containing H C I I d are classified histochemically as type IIB fibres (Termin et al. 1989a & b), it is suggested that there are no changes in the fibre type composition in the
Table 2. Postnatal changes in myosin heavy chain (MHC) isoform of rat extensor digitorum longus (EDL) and soleus muscles
Postnatal age Muscle
MHC
5 weeks
10 weeks
20 weeks
1 year
2 years
1.4 f0.4 10.8 k 2.2 25.7k1.8 62.1 f 4.3 84.7f3.1 15.3f3.1 0
1.7k0.2 12.1 f 1.3 31.3+ 1.2 55.1 k 2 . 5 83.8 f 2.2 16.2 f2.2 0
0.7f0.1 8.2k0.5 35.2k2.1 55.9f2.1 96.9 f2.1" 3.1 f2.1" 0 0
1.2 k0.2 12.1 f 1.8 40.5 f 1.2" 46.2f 1.3" 97.0 f0.8" 3.0 f0.8*
0.5 f O . l 6.0 k 0.8 48.1 f 3.4" 45.4f3.7" 99.9 f0.04" 0.1 f0.04" 0 0
0
0
Values are means+SE; n = 5. " Statistically different (P < 0.05) from 5 weeks.
0 0
422
T. Sugiura et al.
EDL muscle with age. Consequently, the observed MHC changes with age in this study support the previous studies on the postnatal changes in histochemical fibre type composition of rat skeletal muscles. T h e main finding o f t h e present stud!- is that the percentage of H C I I b decreases with a concomitant increase in the percentage of H C I I d in the EDI, muscles with age. Larson 8( Salviati (1989) reported that XlHC composition did not differ significantly between young and old rats in either the EDL or solcus muscles. T h e discrepanc!- between their result and ours may be eyplained b!- the difference in the gel composition and the muscle samplc for \IHC isoform analysis ;for the determination o f I I H C isoforms, the!- used chemicall!- skinned single fibres and O o l l S I X gel containing lo",, glycerol. ( > n the mechanisms underlying the decreased speed of contraction in old age, Idarson (3; Salviati (1989) suggested that probable factors in fast-twitch muscle fibre are an impairment of intrinsic sarcoplasmic reticulum (SR) function and a decrease in SR volume, whereas those in slou-twitch muscle fibre are one or more other mechanism apart from the SR properties. The>also concluded that the alterations either in the myosin heavy chains or in fibre tl-pe proportions \$ere not dominant factors. However, it n a s shown that the intrinsic speed of shortening of muscle fibres lvas direct]!- correlated to actinactiwted myosin .4TPase activity (Bariny 1967) and that the activit!- was basically determined b!thc trpe of XIHC (U-agner 1981, Sivaramakrishnan S. Burke 1982, Reiser t t t i / . 1985). There has been agreement that the myosin .YI'Pase activitl- decreases in the process of ageing in fast-twitch muscle (Gutmann (3; Syo1-i 1971) or in both fast-twitch and slow-twitch muscles (Ermini 1976). Schiaffino rt a/.(1988) have reported that 2X-MHC, which is considered to be identical to H C I I d ('Termin et trl. 1089, Schiaffino et a / . 1989, LaFramboise rr/. 1990). i s the major MHC in the soleus muscle which is transformed b!- high-frequency chronic stimulation. 2X-MHC also correlates with a velocity of shortening N-hich is intermediate between that of slow muscles having almost only H(:I and that of fast muscles containing predominantly- HCI Ib. These findings suggest that the contraction speed of a fibre containing H C I I d is slower than that of a fibre Containing HCITh. I n this respect, Bottinelli ct a / . (1991j
more recently reported that mean maximum velocity of shortening (V,) in type 2X fibres containing MHC-2X was lower (1.45 i 0.066 1 s-l) than that in type 2B fibres (1.80+_ 0.109 1 s-') and type 2A (1.396 0.084 1 s-') and 2X fibres had similar mean values, though type 2 fibres had overlapping of 6.Therefore, it may be considered that the changes in MHC isoforms observed in this study partly account for the reduced myosin ATPase activity and the reduced speed of contraction during the ageing process in both fast- and slow-twitch muscles.
5
\Ye sincerely appreciate the many valuable comments kindl!- given by Professor T. Yoshioka of St A'larianna Lni\-ersit!- School of hledicine and M r S. hlorita of 1-amaguchi University.
R E FE RE N CE S B ~ R 4, , & Ptl..It, D. 1988. Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett 235, 1-53 -15.5, B ~ R ~ x Jl. Y , 1967. ATPase activity of myosin correlated with speed of' muscle shortening. .? Gm Ph18.d 50, 197-218. BILLETER, R., ~ < E I Z M A , C.W., IIOWALD, H. & JESSY, E. 1081. Analysis of myosin light and heavq chain t g m in single human skeletal muscle fibers. I k r 3 Riorhem 116, 389-395. ~ O T T I S E I . I . I ,R., SCHIAFFINO, S. & REGGIANI, c:. 1991. Force -velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. 3 Ph.ysiol (Lond) 137, 655-672. BROOU.:,\ l . I I . & KAISLR, K.K. 1970. Three 'myosin adenosine triphosphatase' systems : the nature of their pH lahilit!- and sulfhydryl dependence. ilistni-hem Cyiochetn 18, 67&672. ~)45IF:I.I-BETTO, D., ZERBATO, E. & BETTO,K. 1986. Thpe I , 2.\ and ZR myosin heavy chain electrophoretic anal?-sis of rat muscle fibers. Biochem Riophlts RESC ~ V Z V138, Z Z981-987. ~~ EI)I)IXGF.R, T.J., h4oss, R.L. & CASSENS, R.G. 1985. Fiher number and type composition in extensor digitoruni longus, soleus, and diaphragm muscles \\ ith ageing in Fisher 344 rats.3Histochem Cylor.hrm 33, 1033-1041. EDSTROSI,I,. & IARSSOS, L,. 1987. Effects of age on contractile and enzyme-histochemical properties of fast- and slow-twitch single motor units in the rat. 3 Ph.~siol( L o n d ) 392, 129-145. ERIIIXI,\I. 1976. Ageing changes in mammalian skeletal muscle. Biochemical studies. Gerontology & SYROVY,I. 1974. Contraction properties and myosin-ATPase activity of fast and slow senile muscles of the rat. Gerontology 20, 239-244.
Ageing rat myosin heavy chain
423
ISHIHARA, A. & ARAKI,H. 1988. Effects of age on the from adult rabbit soleus muscles is correlated with myosin heavy chain composition. 3 B i d Chem 260, number and histochemical properties of muscle 9077-9080. fibers and motoneurons in the rat extensor digiSCHIAFFINO, S., AUSONI,S., GORZA,L., SAGGIN, L., torum longus muscle. Mech Ageing Dez. 45,213-221. GUNDERSEN, K. & LBMO, T. 1988. Myosin heavy KELLY,A.M. 1983. Emergence of specialization in chain isoforms and velocity of shortening of type 2 skeletal muscle. In L.D. Peachey & R.H. Adrian skeletal muscle fibres. Acta Physiol Scand 134, (eds), Hundbook of Physiology, Section 10: Skeletal 575-576. Muscle, pp. 507-537. American Physiology Society, SCHIAFFINO, S., GORZA,L., SARTORE, S., SAGGIN, L., Bethesda. K. & AUSONI,S., VIANELLO,M., GUNDERSEN, KUGELBERG, E. 1976. Adaptive transformation of rat LBMO, T. 1989. Three myosin heavy chain isoforms soleus motor units during growth : histochemical in type 2 skeletal muscle fibres. 3 Musr Res Cell and contraction speed. 3 Neurol Sci 27, 269-289. Motil 10, 197-205. I.AFRAMBOISE, W.A., DAOOD, M.J., GUTHRIE,R.D., SIVARAMAKRISHNAN, M. & BURKE,M. 1982. The free MORETTI,P., SCHIAFFINO, s. & ONTELL, M. 1990. heavy chain of vertebrate skeletal myosin subElectrophoretic separation and immunological idenfragment 1 shows full enzymatic activity. 3' Biol tification of type 2X myosin heavy chain in rat Chem 257, 1102-1 105. skeletal muscle. Biochim Biophys Acta 1035, 109SUGIURA, T . & MURAKAMI, N. 1990. Separation of 112. myosin heavy chain isoforms in rat skeletal muscles LARSSON, L . & EDSTROM,L. 1986. Effects of age on by gradient sodium dodecyl sulfate-polyacrylamide enzymehistochemical fibre spectra and contractile gel electrophoresis. Biomedical Res 1I , 87-91. properties of fast- and slow-twitch skeletal muscles TERMIN, A,, STARON, R.S. & PETTE, D. 1989a. in the rat. 3 Neurol Sci 76, 69-89. Myosin heavy chain isoforms in histochemically LARSSON, I,., EDSTROM, L., LINDEGREN, B., GORZA,L. defined fiber types of rat muscle. Histochemistry 92, & SCHIAFFINO, S. 1991. MHC composition and 453457. enzyme-histochemical and physiological properties TERMIN, A,, STARON,R.S. & PETTE, D. 1989b. of a novel fast-twitch motor unit type. Am 3Physiol Changes in myosin heavy chain isoforms during 261, C93-C101. chronic low-frequency stimulation of rat fast LARSSON, L. & SALVIATI, G. 1989. Effects of age on hindlimb muscles. A single-fibre study. Eur 3 calcium transport activity of sarcoplasmic reticulum Biochem 186, 749-754. in fast- and slow-twitch rat muscle fibres. 3 Ph.ysio1 WAGNER, P.D. 1981. Formation and characterization of myosin hybrids containing essential light chains (Lond) 419, 253-264. REISER,P.J., Moss, R.L., GIULIAN,G.G. & GREASER, and heavy chains from different muscle myosins. 3 B i d Chem 256, 2493-2498. M.L. 1985. Shortening velocity in single fibers
Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat T. S U G I U R A , H. MATOBA*, H. MIYATA, Y. K A W A I and N. M U R A K A M I t Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, * Faculty of Integrated Arts and Sciences, Tokushima University, Tokushima and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
+
SUGIURA, T., MATOBA, H., MIYATA, H., KAWAI, Y. & MURAKAMI, N. 1992. Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiol Scand 144, 419423. Received 26 August 1991, accepted 3 December 1991. ISSN 0001-6772. Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, Faculty of Integrated Arts and Sciences, Tokushima University, Tokushima, and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan. Using gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis, myosin heavy chain (MHC) isoforms were studied in the extensor digitorum longus (EDI,) and the soleus muscles of male Wistar rats at different ages (5, 10, 20 weeks, 1 and 2 years). In the EDL muscle, four types ofMHC isoforms were observed in all age groups. There was an increase in the percentage of HCIId and a concomitant decrease in the percentage of HCIIb with increasing age. No significant difference was observed in the percentages of HCI and HCIIa isoforms in all the age groups. In contrast, the soleus muscle contained two MHC isoforms, HCI and HCIIa. There was an increase in the percentage of HCI and a concomitant decrease in the percentage of HCIIa with increasing age. These results suggest that age-related changes in the MHC isoforms in both the fast-twitch EDI, and the slow-twitch soleus muscles are one factor underlying the age-related decrease in the speed of muscle contraction. Key words : ageing, fast-twitch muscle, myosin heavy chain isoforms, rat, slow-twitch muscle.
I t is well known that the speed of muscle contraction decreases during the ageing process (Gutmann & Syrovy 1974, Larsson & Edstrom 1986, Edstrom & Larsson 1987, Larsson & Salviati 1989). A decrease in the proportion of the fast-twitch fibres is considered to be one possible mechanism underlying the age-related decrease in the speed of contraction (Kugelberg 1976, Eddinger et a/. 1985, Larsson & Edstrom 1986, Edstrom & Larsson 1987, Ishihara & Araki 1988). However, this is not a consistent phenomCorrespondence : Takao Sugiura, Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi 753, Japan.
enon of ageing in all muscles; it is generally reported that the slow-twitch soleus muscle shows an age-related decrease in the relative proportion of fast-twitch fibres (Kugelberg 1976, Eddinger et al. 1985), whereas the fast-twitch extensor digitorum longus (EDL) muscle does not show such alterations (Eddinger et a / . 1985, Larsson & Edstrom 1986, Ishihara & Araki
1988). T h e histochemical actomyosin myofibrillar adenosine triphosphatase (ATPase) reaction of muscle fibres is determined by the difference of myosin heavy chain (MHC) isoforms (Billeter et al. 1981, Danieli-Betto et al. 1986). Recently, a new fast MHC isoform was found in most fast-
419
420
7'.Siigiura
et al.
T a b l e 1. Body and muscle weights of animals at five postnatal periods Postnatal age
nod!
1st
(g)
EI)I. \\t (mg) Soleus u t ( m g ) \ alue5 are means
. irieek5
10 \seeks
20 necks
1 !ear
2 years
144.0k 2.; 66.8 1 .2 57.4+0.7
317.4k8.2 1 5 4 . 0 i 5.9
497.2 k 9.9 211.3kY.5 189.9 7.9
6.53.6k 11.5 279.2 i 1 .0 2 3 7 . i i 10.1
6.50.8k3.5..5 230.8 k 16.9 186.0f 18.0
*
140.9
*
0.9
+SE, N = 5
tv itch muscles using gradient sodium dodecyl sulphate-.polvacrylamide gel electrophoresis (SDS---P.qGE;Bar & Pette 1988). This isoform was tentativel!- designated as H C I I d , since it was detected prcdominantl!- in the adult rat diaphragm. Similarh, Schiafino rt al. (1989) reported the existence of a third MHC, designated as 2X-MHC by the use of monoclonal antibodies specific for MHC. T h e fact that these \lHC isoforms are identical has been suggested by inimunoblot analysis (LaFramboise rt (11. 1990). I t is also suggested that these isoforms ha\-c intcrmediate features between the HCIIb and H C I I a isoforms (Termin rt a / . 1989a & b, Larsaon et ul. 1991). This leads to the assumption that the relative concentration of H C I I d or 2XVHC increases as the speed of contraction decreases during the ageing process. C'nfortunately, to date no information is a\-ailable concerning this. Therefore, the present study was undertaken to clarify the changes with age of the MHC isoforms, including HCIId, in fastand slow-twitch muscles of the rat,
41.A 'YE R I -4 I, S .4 ND 11F: 'I' H 0 D S :Jnimals. These experiments followed the guidelines for thc care and use of animals in the field of ph! siological sciences (The Physiological Society of Japan). Five groups of five male IVistar rats, aged 5, 10, 70 weeks, 1 and 2 years, w r e used in this stud!(see Table 1). They were purchased from K y d o Co. I,td (Saga, Japan). The animals were killed bl- ether inhalation and weighed. After weighing, the soleus and EDI, muscles were r e m o d , and were quickly dissected frec of fat and tendon. The dissected muscles were weighed and frozen in isopentane precooled in liquid nitrogen. They were then stored at -80 "C: until the analysis of XIIIC isoforms was conducted. . y u w p [ ~prc'plrrcrfion. ~ Using a glass homogenizer, a portion of the muscle was homogenized in 10 \-ol of
ice-cold buffer containing 0.1 M KCI, 1 mM E D T A and 20 m \ i Tris-llaleate buffer (pH 7.0). T h e homogenate itas centrifugcd at 1000 g for 10 niin at 4 '(;, and the pellet a a s rehomogenized (1 :40, w/v) in an SDS sample buffer containing 30"" (v/v) glycerol, 5 O , , (v/\-) /I-mercaptoethanol, 2.3 "() (\v/v) SDS, 62.5 m u Tris-HC1 (pII 6.8) and 0.05", (w/v) bromophenol blue. The homogenate was incubated for 10 niin at about 60 "C, and further diluted 1 : 2.5 with the same bufler. Then a 7.5/ t l portion of the homogenate was electrophoresed. SDS-P.4GE. T h e analysis of M H C isoforms was electrnphorcticalll- carried out according to the method of Bar & Pette (1988) as modified by Sugiura & llurakami (1990). Briefly, SDS-P.4CiE was performed on a slab gel (7 x 9 cm x 1 mni) using a S4",,, ( w / v ) polya~rylarnide/3(WO",~ (v/v) gly-cerol gradient separating gel and a 3.j0,, (w/v) polyacrylamide stacking gel containing 35 "(,(v/v) glycerol. Electrophoresis w s first run at 50 V until the tracking dye cornpierel>- entered the separating gel, and then at 1.50 V for about 18 h at 8 "C. Gels were stained in a solution containing 0. 1 O 0 ( w / v ) Coomassie brilliant blue, 50c1(,(../I) methanol, and lo",, (v/v) acetic acid, and destained b!- diffusion in a solution of S",, ( v / v ) methanol and 7",, (\-/\-) acetic acid. T h e percentage distribution of MI I C isoforms was calculated b!- the use of a Shimadzu CS-930 TLC Scanner (Kyotn, Japan) equipped with a laser source attachment (ISA9000). S/o/rs/ic.s. Means and standard errors were calculated from indi\ idual values by standard procedures. A one-nay anall-sis of variance (ANOVA) was performed in order to evaluate the significance of ageassociated trends. Tukey's method was used for intergroup comparison. The 0.05 confidence level was chosen for statistical significance.
RESULTS Figure 1 and Table 2 show the electrophoretogram and the percentage of MHC isoforms in the EDI, and soleus muscles, respecti\ely. XIHC isoforms of the EDJ, muscle
Ageing rat myosin heazy chain
421
( P < 0.05) and the percentage of H C I I a was significantly lower (P < 0.05) compared with the 5-week-old soleus muscle. DISCUSSION Fig. 1. Electrophoretograms of myosin heavy chain (MHC) isoforms from the rat extensor digitorum longus (EDL) and soleus muscles. Lanes: 1, 5-weekold EDL; 2,2-year-oId EDL; 3, 5-week-old soleus; 4,
2-year-old soleus. Only the area of the MHC region is shown.
are separated into four types, HCIIa, H C I I d , H C I I b and HCI, in order of increasing electrophoretic mobility, on a 5-80/, gradient SDSPAGE (Termin et al. 1989a & b). There was no change in the percentage of HCI and HCII (IIb, I I d and IIa) isoforms in the EDL muscle from the 5-week to the 2-year age groups. However, there were small changes in the percentages of HCII isoforms with increasing age. T h e percentage of H C I I d was significantly higher at 1 and 2 years than at 5 weeks, whereas the percentage of H C I I b was significantly lower (P< 0.05) at 1 and 2 years than at 5 weeks. No significant difference was observed in the percentage of H C I I a between the age groups. I n contrast to the E D L muscle, the soleus muscle contained only two MHC isoforms, HCI and HCIIa, at 5 weeks of age, HCI being more prominent than HCIIa. Twenty weeks later, the percentage of HCI was significantly higher
Histochemical studies using rat skeletal muscles have shown that in the soleus muscle the percentage of type I fibres increases during development (Eddinger et al. 1985), whereas in the EDL muscle fibre type differentiation occurs by approximately 3 weeks postpartum (Kelly 1983), and the transformation of muscle fibres from type I1 to type I does not occur during subsequent development (Eddinger et al. 1985, Larsson & Edstrom 1986, Ishihara & Araki 1988). T h e histochemical myofibriliar ATPase reaction of a muscle fibre is determined by its myosin heavy chain composition (Billeter et al. 1981, Danieli-Betto et al. 1986). Recent reports (Termin et al. 1989a) have also shown that type I I D fibres containing H C I I d are almost indistinguishable from type I I B fibres containing HCIIb, using the criteria of Brooke & Kaiser (1970). I n this study, as shown in Table 2 , there was an increase in the percentage of HCI and a concomitant decrease in the percentage of HCIIa with increasing age in the soleus muscle. O n the other hand, in the E D L muscle, there were no changes in the sum of H C I I b and H C I I d from the 5-week to the 2-year age groups. Assuming that fibres containing H C I I d are classified histochemically as type IIB fibres (Termin et al. 1989a & b), it is suggested that there are no changes in the fibre type composition in the
Table 2. Postnatal changes in myosin heavy chain (MHC) isoform of rat extensor digitorum longus (EDL) and soleus muscles
Postnatal age Muscle
MHC
5 weeks
10 weeks
20 weeks
1 year
2 years
1.4 f0.4 10.8 k 2.2 25.7k1.8 62.1 f 4.3 84.7f3.1 15.3f3.1 0
1.7k0.2 12.1 f 1.3 31.3+ 1.2 55.1 k 2 . 5 83.8 f 2.2 16.2 f2.2 0
0.7f0.1 8.2k0.5 35.2k2.1 55.9f2.1 96.9 f2.1" 3.1 f2.1" 0 0
1.2 k0.2 12.1 f 1.8 40.5 f 1.2" 46.2f 1.3" 97.0 f0.8" 3.0 f0.8*
0.5 f O . l 6.0 k 0.8 48.1 f 3.4" 45.4f3.7" 99.9 f0.04" 0.1 f0.04" 0 0
0
0
Values are means+SE; n = 5. " Statistically different (P < 0.05) from 5 weeks.
0 0
422
T. Sugiura et al.
EDL muscle with age. Consequently, the observed MHC changes with age in this study support the previous studies on the postnatal changes in histochemical fibre type composition of rat skeletal muscles. T h e main finding o f t h e present stud!- is that the percentage of H C I I b decreases with a concomitant increase in the percentage of H C I I d in the EDI, muscles with age. Larson 8( Salviati (1989) reported that XlHC composition did not differ significantly between young and old rats in either the EDL or solcus muscles. T h e discrepanc!- between their result and ours may be eyplained b!- the difference in the gel composition and the muscle samplc for \IHC isoform analysis ;for the determination o f I I H C isoforms, the!- used chemicall!- skinned single fibres and O o l l S I X gel containing lo",, glycerol. ( > n the mechanisms underlying the decreased speed of contraction in old age, Idarson (3; Salviati (1989) suggested that probable factors in fast-twitch muscle fibre are an impairment of intrinsic sarcoplasmic reticulum (SR) function and a decrease in SR volume, whereas those in slou-twitch muscle fibre are one or more other mechanism apart from the SR properties. The>also concluded that the alterations either in the myosin heavy chains or in fibre tl-pe proportions \$ere not dominant factors. However, it n a s shown that the intrinsic speed of shortening of muscle fibres lvas direct]!- correlated to actinactiwted myosin .4TPase activity (Bariny 1967) and that the activit!- was basically determined b!thc trpe of XIHC (U-agner 1981, Sivaramakrishnan S. Burke 1982, Reiser t t t i / . 1985). There has been agreement that the myosin .YI'Pase activitl- decreases in the process of ageing in fast-twitch muscle (Gutmann (3; Syo1-i 1971) or in both fast-twitch and slow-twitch muscles (Ermini 1976). Schiaffino rt a/.(1988) have reported that 2X-MHC, which is considered to be identical to H C I I d ('Termin et trl. 1089, Schiaffino et a / . 1989, LaFramboise rr/. 1990). i s the major MHC in the soleus muscle which is transformed b!- high-frequency chronic stimulation. 2X-MHC also correlates with a velocity of shortening N-hich is intermediate between that of slow muscles having almost only H(:I and that of fast muscles containing predominantly- HCI Ib. These findings suggest that the contraction speed of a fibre containing H C I I d is slower than that of a fibre Containing HCITh. I n this respect, Bottinelli ct a / . (1991j
more recently reported that mean maximum velocity of shortening (V,) in type 2X fibres containing MHC-2X was lower (1.45 i 0.066 1 s-l) than that in type 2B fibres (1.80+_ 0.109 1 s-') and type 2A (1.396 0.084 1 s-') and 2X fibres had similar mean values, though type 2 fibres had overlapping of 6.Therefore, it may be considered that the changes in MHC isoforms observed in this study partly account for the reduced myosin ATPase activity and the reduced speed of contraction during the ageing process in both fast- and slow-twitch muscles.
5
\Ye sincerely appreciate the many valuable comments kindl!- given by Professor T. Yoshioka of St A'larianna Lni\-ersit!- School of hledicine and M r S. hlorita of 1-amaguchi University.
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