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Skeletal muscle regeneration in young rats is dependent on growth hormone Michael Uliman and Anders Oldfors I)epartment or Pathology. I:nit er.wty (~t (;ottu'nbur~,. Sahlgrcn "s Ito.~pital. S-41345 (;iitcborg ¢,S'wcth.p~ (Rcceivctl 14 Januap... ttJgl) (Revised. received 21 June. 1~t91 ) (Accepted 26 Jullc, It,l'41 )

Key words: Skeletal muscle: Regeneration: Notcxin; Satellite cells: Growth hormone: Hypophysectomy: l'hymidinc; Rats Summary Skeletal muscle fibres have a well known ability to regenerate after different kinds of injury. This stutb was undertaken to establish if regenerating skeletal muscle is dependent on growth hormone (GH) in the same manner as normal, growing skeletal muscle in young rats. Muscle regeneration was achieved by injection of notexin into the soleus muscle. Initial necrosis, which included all muscle fibres, was followed by a rapid and uniform regeneration throughout the muscle. ('ell proliferation was estimated by scintillation counting and autoradiography of incorporated [3H]thymidinc, injected intravcnou,,,ly 1 h bcforc killing. 7 or 27 days after the initiation of regeneration. GH dcficicncy was accomplished by hypophyscctom~ 4 days before the [3H]thymidinc injection. Cell proliferation was diminished in both regenerating and normal muscle of the hyl~physcctomizcd rats compared to control and GH-substitutcd rats. After 7 days of regeneration thc rcduction of cell proliferation sccn in hypophyscctomizcd rats was less pronounced in the regenerating than in the normal muscle. These findings demonstrate that GH plays an important role for muscle regcneration in young rats, although other substances appear to bc of greater importance during the carly stages of regeneration.

Introduction Skeletal muscle fibres develop from embryonic. myogenic cells, which fuse to form multinucleated myotubeg~ and subsequently develop into muscle fibres (Fischman 1986). Another course of the myogenic cells is to develop into satellite cells, located underneath the basal lamina of muscle fibers. In contrast to the nuclei of the muscle fibres, the satellite cells continually divide to supply the growing muscle fibers with new myonuclei during development. In the mature muscle, the satellite cells are dormant, but may act as a stem cell pool for muscle repair (Mazanet and FranziniArmstrong 1986). The necessity of G H for the normal postnatal development of skeletal muscle is well established (Cheek and Hill 1975; Cheek 1985; Fiorini 1987). During the fetal and neonatal periods, skeletal muscle develops

Correspondence to: Dr. Michael UIIman. D e p a r t m e n t of Pathology'. University of G o t h e n b u r g , Sahlgren's Hospital. S-41345 G6teborg, Sweden. Tel.: 31 862 084; Fax: 31 274 208.

independently of growth hormone (Florini 1987), but in neonatally hypophysectomized rats, body- and musclegrowth declines gradually and almost ceases at about 4 weeks of age (Walker et al. 1950; Glasscock ct al. 1990). The mode of action of G H on skeletal muscle is unclear, but circulating and locally produced IGF-I appears to act as an intermediary growth factor of great importance (Sara and Hall 1990). Skeletal muscle fibres have a well recognized ability to regenerate after necrosis, and the source of the new muscle fibers is the satellite cells (Church et al. 1966; Snow 1978; Mazanet and [:ranzini-Armstrong 1986). This ability to regenerate is essential in many myopathies, after different kinds of muscle trauma and transplantation of free muscle grafts. It is also fundamental for the treatment of primary myopathies with myoblast transfer (Partridge et al. 1990). After necrosis of muscle fibres, satellite cells arc stimulated to multiply and fuse to form myotubes and new muscle fibres in a manner similar to normal muscle development. The mechanisms that initiate and regulate satellite cell proliferation and differentiation during muscle regeneration arc still not known, although a number of

6N factors arc known to influcnce growth of muscle cells in vivo ;.is well as in vitro: Growth of the postnatal skeletal muscle is rcportcd to be rcgulatcd by factors like age (Schultz and IApton 1982), nutrition (()Idfors ct al. 1083: Chcck 1985). physical activity (Schuhz 10Nt)), musclc stretch (Matthews et al. 19911). insulin, scx hormones, thyroxine (Cheek 1985) and insulin-like growth factors (Sara and lhill 1990). A number of substances have bccn shown to modulatc multiplication and differentiation of myogenic cclls in vitro: e.g. l'ibroblast growth factor (FGF). transforming growth factor-/3 ('l(iF-lJ) (AIIcn and Boxhorn 198c)). and insulin-like growth factor I and II (IGF-I and IGF-II) (l)odson cl al. 1985). A polypcptMe dcrivcd frolll crushed skeletal muscle has been shown to stimulate satcllitc ccll prolifcration both in vitro and in vivo (l'lischoff lqg6l. l h e effect of G H on myogcnic cells in vitro is unclcar. Apart from thc stimulation of diffcrcntiation found in one spccific cell line (Nixon and Green 1984), no effect of growth hormone on cultured myoblasts in vitro has been dcmonstrated (Ewton and Florini 1981: Allcn ct a[. lC,~N3). These studies, however, were rnade on cclls thal morc closely rcsemble embwonic than maturc muscle cclls (Florini 1987). Furthcrmorc, thc satellite cell may alter its characteristics when dissociatcd from the mature muscle fibrc for cultivation in vitro (Allen and Rankin 1990). Scvcral papers havc been published on the effects of G H on muscle prcl)arations in vitro. Incubation with G l i of diaphragm of hypophyscctomizcd rats did not affcct thc incorporalion of tritiated thymidinc ([31t]d'l'hd)((hfldspink and (hfldberg 1975). On thc other hand, amino acid up-take and protcin synthesis of diaphragm muscle of hypophyscctomizcd young rats was increased by direct stimuhition of (iH in vitro (Albcrsson-Wikland and Isaksson 1976). Ahhough growth hormone (GH) is essential for normal postnatal cell prolifcration and growth of skeletal muscle, little is known about the importance of G H for rcgcncrating muscle. Administration of (;H to norreal aduh r;tts. 4 months old, cnhanccd weight and I)NA content in both normal muscle and muscle rcgcncrating aftcr ischcmic necrosis (Ullman ct al. 1989: L;llman and ()ldfors 1989). However, hypophyscctomy did not significantly reduce the incorporation of [ ' l l ] d T h d in rcgencrating muscle of rats of thc samc age (Sommerland ct al. 1989). In sencsccnt rats, 25 months old. G t l trcatmcnt for 10 weeks did not significantly increasc the weight of regenerating muscle (l, Jllman ct al. 19911). Several met[lods have been described for induction of necrosis with enstiing regeneration of skeletal muscle. Wc have hitherto applied the wcll established method of inducing necrosis by cutting the supplying vessels and ncrvcs of the muscle (Carlson and (}titmann l t)75). This causes an ischemic necrosis cngaging

the central parts of thc muscle but spares periphcral muscle fibres and satellite cells. Thc regeneration is accordingly non-uniform, ;.is it starts from thc pcripht i t and progrcsscs centrally ((',lrlson lqg]). In this study we havc used intramuscular in.icction of notcxin, as it induces a complete nccrosi.,, of :ill musclc fibres of the solcus muscle, but spares the satcllitc cells as v~,cll its the blood circulation (llarris 1988). This would facilitate a more unilornl and laslcr regeneration than that achieved by the ischcrnic mcthod. Apparently thc sensitivity for (lit is low in oldcr rats, but morc pronounced ill adult rats aild accordingly the prcseni stud}, was undertaken to hwcstigatc if the effect of G H deficiency on muscle rcgcncrathm in young rats is even more prontRlnced. The age of g wccks was choscn as rats of this :lgc arc still rapidly growing and the growth is high[.x dcpcndcnt on (}tl.

Material and methods Animals

Male Spraguc-I)awlcy rats wcrc purchascd from ALAB (Stockholm, Sweden) arm kept in phtstic cagcs in a room with artificial light {from 6 a.m. to 6 p.m.), constant temperature (24 ...I. Zapl ++I(+ ,Schciv, illcr and [-.R. I:roc~,uh (It.~8X) Rccoml+'inant hulllan in~,ulin-likc growth t'actor I stinl)llatv's grt+V~.lh and h;ts di,,tillCl cfl¢¢ts tm t)rg;lll >,J],..' ill Ilypc~physectt'~nlJzcd i';lls. PI'OC. N a i l . . \ c a d . ScJ. [ 'SA. 85:4S,',:9 .4XtD. llatt~,..I.l'k (lUSSI Nolo\in: il:'., act+ore, cm skeletal muscle. In: I..('. NCIIIIL I{. Nil+el+us and S. l ' h u s l c l t ([-dx.). N e u r o m u s c u l a r Junctl,Ut. I N~.",iCl NCiCllCC t"ul+,li,,hcr ,, ( [:liomcdical l)i',, ision ). ,.\m~,tcrdalll. I+,p. | ~ - 47tL Ilalll,,. I1'~. M \ . h,hllx~.+n and I'. Karlss~m (ItJ75)l)ath~+h,gical IC',I+~t+ID+CS (+l I;1| x k ¢ l c t a l IIltlSCIc tt+ iI ",,ingle ~,UI'IcLII~I11COtlS, ill.ICetit+rl t+l ;I toxin i,,t+l+it¢,,lIll+I+1 the venom ~I the Auslrahan tiger ~ll~lkC. \ + ~ h ' ( / l l ~ "+( I I l + ( l l l ~ ",+tll(l{lt'~. ('llll. l { \ p . Phil1"111. Phys.. 2: +;.. g,,:llC tlLIIl,,IcclJt+I1. IIlt+J¢ctlJal r+._'palr nlcChilnJMil:,,+ .I. Ncurol. Sci.. t)X C';uppl.l: 12U. Sara. \".l,I. and K. l lall (199()) ]i+i,,tdmu.likc grov, th f~ictt+l,, and th,+.'il bindirlg prt+tcins. Ph.~,iol. Ru:+,.. 7II: 5"31 ++,II. ,'qchuh,,. I{. (It~St+) Sale+life ,,+ell l+,cha',ic,r dttring sk,,:lctal nmsulu gr,.r,.'+,thand r,.:gcneruti,.m Md S,,.'i.~p,utls [ixcrc.. 21

Skeletal muscle regeneration in young rats is dependent on growth hormone.

Skeletal muscle fibres have a well known ability to regenerate after different kinds of injury. This study was undertaken to establish if regenerating...
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