Rat triceps surae was minced and orthotopically autografted. Twitch time to peak, maxima tetanic tension, lactate dehydrogenase activity and total creatine concentration were measured in muscles regenerating for 30, 60 and 90 days. If the minces were frozen and thawed before grafting, muscle regeneration was suppressed. If they were further heated before grafting, muscle regeneration was also suppressed. If one half of the mince was either frozen and thawed or frozen, thawed and heated, and then recombined with the remaining half, muscle regeneration was delayed. However, at 90 days, 'intensive properties' (twitch time to peak, maximum tetanic tension, total creatine concentration and lactate dehydrogenase activity) of regenerates obtained from such partially treated minces were similar to those of regenerates obtained from untreated minces although their 'extensive properties' (weight and maximal tetanic force) were approximately halved. The extent of regeneration depends on the mass of untreated mince autografted and thus, presumably, on the number of viable muscle stem cells initially present in the mince.

Native myosin isozymes of rat muscles have been isolated by electrophoreses in non-dissociating conditions. Their mobilities were measured, using taenia coli myosin as an internal standard and their relative concentrations were determined by computer planimetry of the electrophoretograms. Three isozymes were observed in extensor digitorum longus (EDL), two in soleus (SOL), four in neonatal muscles and four in muscles three days before birth. Regenerates of minced EDL or SOL muscles in adult animals had no native myosin the third day after surgery; they were similar to neonatal muscles 15 days after surgery and to adult muscles 60 days after surgery.Muscle myosin is an hexameric molecule in its native state. Various isozymes, differing by their heavy-chain primary structures or by the type of their alkaline light chains, can be separated in their native state by electrophoreses in nondissociating conditions [I -41. Slow muscle myosin can thus be separated from fast muscle myosin [I, 21. Neonatal [5, 61 and embryonic [6] isomyosins have also been separated by this technique, whilst their chemical individuality has been supported by immunochemical methods or peptide mapping after enzymatic cleavage of the myosin heavy chains. Using taenia coli myosin as an internal standard, we are able to measure accurately RF values for the various isoforms of myosin in adult, neonatal and embryonic muscles and, using an interactive computer-controlled scanning device, to measure the relative proportions of the various isoforms.Adult muscles regenerating from a local cold injury express transiently fetal myosin heavy chains [7]. We have therefore analysed the isomyosins of regenerating muscles, using a model described by Carlson [S] in which the muscle is minced and orthotopically autografted. After an initial phase of degeneration and fragmentation of the graft, regeneration proceeds, recapitulating ontogeny. 15 days after surgery, the regenerate is contractile [9] and partially innervated [lo]. 60 days later, the fibres are hypertrophied [S], their tension may be back to normal [Ill, their force-velocity is similar to that ofthe original muscle [I21 and the functional recovery is satisfactory [ll]. We show in this work that the isozymic transition characteristics of muscle growth from embryo to adult stages [6] also occur in adult rat during muscle regeneration. MATERIALS AND METHODSStriated muscles excised from rats (Wistar, outbred local stock, obtained from the Katholieke Universiteit te Leuven) were Abbreviations. SOL, adult soleus muscle; EDL, adult extensor digitorus longus muscle; FM,, FM, and FM,, myosins of adult fast muscle 1,2 or 3; SM;, SM, and SM,, myosins of adult slow muscle l', 1 or 2; fM,, fM,, fM,, fM, and fM,, myosins of neonatal muscle 1,2, 3,4 or 5 ; eM,, eM,, eM, and eM,, myosins of embryonic muscle, 1,2, 3 or 4; R,, ratio of the electrophoretic mobility of myosin to the mobility of an internal standard.Enzyme. Myosin ATPase (EC 3.6.1.3).used. Embryonic muscles (symbolized 'eM') were obtained by careful...

Right triceps surae of 3-week-old Wistar rats were minced and devitalized with liquid nitrogen, a treatment which completely inhibits their ability to regenerate when they are orthotopically autografted. In a first series of experiments, cells were isolated from the left triceps surae, mixed with the devitalized right mince and autografted; in a second series, cells were moreover allowed to proliferate in vitro for a few weeks before being grafted. The regenerates were examined 60 days after surgery. In the first series, all the regenerates were contractile and developed a maximal isometric tetanic force of 18 +/- 6 mN (n = 5); they contained 152 +/- 80 muscle fibres located proximally, the number of which decreased along the proximo-distal axis, being 24 +/- 24 in the median part of the regenerate. The muscle fibres appeared histologically normal except for their shortness (less than 10 mm) and narrowness (mean luminal diameter: 30 microns). In the second series, 2 out of 5 regenerates were comparable with those of the first series except that their fibres were shorter; the 3 other regenerates were unexcitable. These experiments demonstrate that cells isolated from an adult striated muscle are able to regenerate striated muscle fibres in an adult animal and that these cells can retain this property if they are grown in culture.

Triceps surae of 3 week-old female Wistar rats were minced and orthotopically autografted. Thirty days later, the regenerates developed a force of 314 +/- 58 mN during an isometric maximal tetanus and the maximal area of the muscle tissue was 3.2 +/- 0.5 mm2 (n = 9) in a cross-section. At 60 days, their mean maximal isometric force was increased (1159 +/- 367 mN) as also were their maximal muscle cross-section area (6.8 +/- 0.7 mm2;n = 6). If the minces were devitalized by a freeze and thaw procedure, the regenerates neither contracted under electrical stimulation nor contained any muscle fibre. If devitalized minces were seeded with myogenic cells isolated from the contralateral triceps surae, orthotopically autografted and allowed to regenerate for 30 days, the regeneration was weak: 46 +/- 14 mN of force, 1.2 +/- 0.3 mm2 of muscle area in minces devitalized by cold (n = 7) and 23 +/- 8 mN of force, 0.2 +/- 0.1 mm2 of muscle area in minces devitalized by cold supplemented by heat (n = 8). If viable minces were seeded with myogenic cells, there was no improvement in the extent of regeneration: 275 +/- 83 mN of force, 2.0 +/- 0.6 mm2 of muscle area at 30 days (n = 7) and 738 +/- 191 mN of force, 5.6 +/- 0.9 mm2 of muscle area at 60 days (n = 5). Consequently, although it is possible to induce regeneration by grafting myogenic cells into a devitalized mince, this procedure has no effect when applied to a viable mince.