Acta Physiol Scand 1991, 142, 523-524
Subtle indications of muscle damage following eccentric contractions J. F R I D E N , R. L. LIEBER* and L.-E. T H O R N E L L ? Department of Hand Surgery, U m e i University Hospital, Sweden; *Division of Orthopaedics and Rehabilitation, Veterans Administration Medical Center and University of California, San Diego, USA and +Department of Anatomy, University of Umeb, Sweden
Intensive training involving eccentric muscle contractions results in muscle fibre damage (FridCn 1984). In a recent paper we have described a selective structural fast-twitch fibre adaptation to eccentric contractions in an animal model (Lieber & FridCn 1988). It is the purpose of this study to further investigate the more subtle indications of cellular reaction following high-tension, fast-twitch-biased eccentric exercise in our rabbit model. The right legs of 10 New Zealand rabbits were immobilized by passing 3.2 mm Steinmann pins through distal femur and proximal tibia and securing them to a rigid frame. The distal tendon of m. tibialis anterior (TA) was attached to a dual-mode servomotor (Cambridge Technology Model 310, Cambridge, MA) and aligned with the translation axis of the motor. The peroneal nerve was isolated and a Dastre electrode pair was placed beneath the nerve for muscle activation. Under computer control (Lieber et al. 1986) muscle length was adjusted to the length at which twitch tension was maximum (Lo), and maximum tetanic tension (Po) measured while stimulating at 100 Hz. Based on our previous architectural studies, muscle fibre length was calculated as 0.67 x Lo.T o obtain cyclic eccentric contractions, the muscle activation pattern from isometric contractions (40 Hz, 400 ms, 0.5 Po, 600 ms relaxation) and passive stretch (25% linear stretch, 400 ms, 0.3 p m half-sarcomere-') were superimposed during 30 min. Following the exercise period, animals were maintained under halothane anaesthesia for 1 h, permitting the early inflammatory response and recovery from short-term fatigue. Animals were then killed and the T A excised. The entire muscle was frozen in isopentane cooled by liquid nitrogen ( - 159 "C) and subsequently cross-sectioned in a Reichert Jung cryostat at - 19 "C. Sections (10pm thick) were Received 11 March 1991, accepted 7 May 1991. Key words: Fibre damage, muscle injury, z-disk. Correspondence : Jan Fridin, M. D., Ph.D., Department of Hand Surgery, University Hospital, S901 85 Umeb, Sweden.
Fig. 1. Micrographs of serially sectioned rabbit TA following cyclic eccentric contractions. Calibration bar = 50 pm. (a) Haematoxylin and eosin, (b) NADH, (c) fibronectin.
stained u ith haeniarosylin and eosin, X.ID1 I, and for demonstration of niyotibrillar .\TPase (Ilubou-itz 19x5). The remaining sections, t ~ pjm thick, were stained \vith monoclonal antibodies specific for slo\r-and fast-twitch LlH(:s according t o kilby & Ilhoot (1988). and with monoclonal fibronectin (kindly provided b!- Dr 1-irtanen. I Ielsinki) according to a modification of the method emplo! ed b!- \-artio and coworkers (1987). Standard indirect perosidase-antiperoxidase (P.4P) were used. P.\P was obtained from Dakopatts, Copenhagen, Denmark. T h e sections \\ere csamined in a I x i t z Ilialux microscope. The H&E staining revealed a significant portion of lightl!- stained, \er! large tibrea (Fig. 1 a). Oxidative staining (TPNI I) demonstrated a IOH mitochondria1 enz\me actibity in these fibres (Fig. 1 b), i.e. significantl! less than in ordinar! t!.pe 2 fibres. Staining with alkaline m!-ofibrillar .-\TPase and monoclonal antibod! specific for fast-twitch XfHCs showed a homogeneous and comparati\-el!- strong intensity ot'the large fibres. In the sections of the large fibres treated with anti-slow-twitch myosin a weak staining was obsened. Though no routine histochemical staining could reveal any more intracellular pathology, the fibronectin staining consistently demonstrated a heavilk- stained core, somewhat eccentrically positioned (Fig. 1 c), -411giant fibres contained these abnormalities, and these changes were confined to rhc large type 2 fibres. i\lso, the cores appeared denser than surrounding myofibrillar components using anti-fast twitch myosin. This study demonstrated the occurrence of more subtle intracellular changes of eccentrically exercised muscles, not generall!. detected by routine histological stainings. While fibronectins are adhesive to highmolecular-weight gl!-coproteins found in basement membranes and interstitial connecti\-e tissue matris, me here report an intracellular deposit. Presentl!-, \\e
assunie these alterations to reflect autophagic vacuolization of membrane components in severely damaged fibres, subsequently undergoing necrosis. It is reasonable to believe that these fibres do not function optimally, but in order to judge the physiological implication of this abnormality its longitudinal distribution as \\ell as its ultrastructural composition has to be further investigated. This nark was supported by the Veterans Administration, N I H grant .4R 40050-02, the Research Council o f the Swedish Sports Federation and the Swedish Society of Medicine.
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