|  | Feature: “ICSEMIS Researchers Award” | No.56 May 2009 |
|  |  | print / save view |

New Aspects on Myofibrillar Remodeling in Human Muscle after Eccentric Exercise Lars-Eric Thornell & Lena Carlsson |
Introduction The myofibrillar and cytoskeletal alterations observed in delayed onset muscle soreness (DOMS) caused by eccentric exercise have generally been considered to represent damage (e.g. Clarkson and Hubal 2002, Proske and Allen 2005). One basis for this assumption is the pioneering work by J Friden (1983) “Exercise induced Muscle Soreness”. Fridén performed ultrastructural studies on biopsies from volunteering students who either performed downhill running or reversed biking, both events leading to eccentric muscle actions that gave rise to substantial delayed onset muscle soreness. He observed that the myofibrillar Z discs, the anchoring structure for the thin actin filaments of subsequent sarcomeres were altered. They were broadened and irregular and it appeared as if the material of the Z discs had become extended into the sarcomeres, giving name to the term Z disc streaming and Z disc disruption. Extended studies on a rabbit model for eccentric exercise have further led to the concept that repetitive active lengthening of skeletal muscle initiates a sequence of events that includes muscle cytoskeletal breakdown, loss of the cytoskeletal protein desmin and loss of cellular integrity as evidenced by sarcolemmal damage leading to inflammation occurs early during heavy eccentric exercise (Friden and Lieber 2001, Lieber and Friden 2002, Lieber et al 2002). Having been in the field of research on desmin and muscle pathology and the development of new tools to study the effects on proteins by eccentric exercise prompted reinvestigation of what happens in human muscle upon exercise leading to DOMS. In a series of papers (Yu and Thornell 2002, Yu et al. 2002, 2003, 2004, Carlsson et al 2007) using the same experimental protocols as in the studies of Friden in the 80s, but using immunohistochemical, transmission and immunoelectron microscopy methods, we showed that in human muscle there is no cytoskeletal degradation of desmin. On the contrary more desmin was present in the form of longitudinal strands at sites where more sarcomeres in parts of the myofibrils were seen. These so-called nonius periods, reflect the addition of sarcomeres, which will give rise to a lengthening of the myofibrils that in turn gives a mechanistic explanation for the second bout effect – that upon additional exercise no DOMS will appear. Furthermore we proved that there was neither sarcolemmal disruption of the extent that fibronectin did not enter the muscle fibres, nor did we observe invasion of inflammatory cells. In the study presented at ICSEMIS 2008, muscle biopsies from persons affected by DOMS after a voluntary eccentric exercise were further analyzed with immunohistochemistry and antibodies to a number of new proteins related to the myofibrillar Z disk and M band. Our aim was to reveal how these proteins are affected by the eccentric exercise and how they might be involved in myofibrillar remodeling and the formation of new sarcomeres. Material and Methods The sample comprised sixteen healthy men with a mean age of 24.3 years of which ten volunteers did a bout of eccentric exercise and six served as controls. Muscle samples were processed for immunohistochemistry. A special methodology was used to obtain semi-thin (0.2 µm) sections, which allows high-resolution immunolocalisation of antigens in myofibrils and the muscle fibre cytoskeleton (See Carlsson et al 2007). Well-characterized antibodies against the following myofibrillar and cytoskeletal proteins were tested: Filamin, FATZ (myozenin; calcsarcin), ZASP (Cypher), telethonin (Tcap) and obscurin. Double labelling was performed with one monoclonal and one polyclonal antibody, followed by secondary antibodies coupled to fluorochromes of different wavelengths. The sections were evaluated in a Nikon eclipse E 800 microscope and a SPOT RT Colour camera was used for image acquisition. Results Myofibrillar alterations corresponding to the ultrastructural hallmark of DOMS, Z disc streaming, were unstained for several Z disc related proteins like: myozenin, ZASP, telethonin, titin, alpha–actinin and nebulin, whereas two newly recognized proteins filamin and obscurin showed a marked increase of staining together with increased staining for desmin and myotilin. Especially the staining for obscurin showed a new and interesting feature. Staining for obscurin was mainly localized to the M band region and was unaffected in small myofibrillar alterations, whereas in large alterations it was co-localized with increased staining for F-actin, myotilin and desmin. Discussion and Conclusion The study explored further the dynamic molecular remodelling that takes place upon eccentric exercise and shows a profound rearrangement of several Z disc related proteins, which leads to the insertion of new sarcomeres and lengthening of myofibrils. Obscurin is here shown to be an important component of an M band related extrasarcomeric cytoskeleton, which encircles the myofibrils and attach them to the sarcolemma. In the small alterations obscurin seems to serve as a scaffold for the rearrangement of the sarcomeres together with the longitudinally aligned desmin intermediate filaments. Our new results are incorporated in a more advanced scheme reflecting the molecular events of human in situ sarcomerogenesis. Importantly the study challenges the concept of DOMS being due to the following events: 1) Mechanical damage to muscle tissue during the exercise (disruption of myofibrillar Z discs, the cytoskeleton with loss of desmin and of the sarcolemma), 2) Leukocyte and macrophage infiltration leading to inflammation. 3) Activation of satellite cells leading to regeneration and repair (Peake et al 2005). It is concluded that experimental models in animals do not mimic what happens in human muscle on eccentric exercise. References Clarkson PM, Hubal MJ. Exercise-induced muscle damage in humans. Am J Phys Med Rehabil. 2002. 81 (11 Suppl):S52-69. Review. Proske, U; Allen, T J. Damage to Skeletal Muscle from Eccentric Exercise. Exercise & Sport Sciences Reviews. 2005. 33(2):98-104, Fridén J, Lieber RL Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components. Acta Physiol Scand. 2001. 171(3):321-6. Review Lieber RL, Fridén J Morphologic and mechanical basis of delayed-onset muscle soreness.J Am Acad Orthop Surg. 2002. 10(1):67-73. Lieber RL, Shah S, Fridén J. Cytoskeletal disruption after eccentric contraction-induced muscle injury. Clin Orthop Relat Res. 2002. 403 Suppl):S90-9. Review. Yu JG, Thornell LE. Desmin and actin alterations in human muscles affected by delayed onset muscle soreness: a high resolution immunocytochemical study. Histochem Cell Biol 2002. 118:171-179. Yu JG, Malm C, Thornell LE. Eccentric contractions leading to DOMS do not cause loss of desmin nor fibre necrosis in human muscle. Histochem Cell Biol. 2002. 118: 29-34 Yu JG, Dieter O Fürst; Thornell LE. The mode of myofibril remodeling in human skeletal muscles affected by DOMS induced by eccentric contractions. Histochem Cell Biol. 2003. 119: 383-393 Yu JG, Carlsson L, Thornell LE. Evidence for myofibril remodeling as opposed to myofibril damage in human muscles with DOMS: an ultrastructural and immunoelectron microscopic study. 2004. Histochem Cell Biol 121:219-227. Carlsson L, Yu JG, Moza M, Carpén O, Thornell LE. Myotilin: a prominent marker of myofibrillar remodelling. Neuromuscul Disord. 2007. 17(1):61-8 Carlsson L, Yu JG, Thornell LE. New aspects of obscurin in human striated muscles. Histochem Cell Biol. 2008. 130(1):91-103. Peake J, Nosaka K, Suzuki K. Characterization of inflammatory responses to eccentric exercise in humans. Exerc Immunol Rev. 2005. 11:64-85. Schematic models of myofibrillar remodelling induced by eccentric exercise. A new sarcomere can be formed either by a split of a single Z-disc(A) or by an alteration of a single sarcomere (B). In the model the major constituents of a myofibril are shown: Myosin (black), actin (dark blue), titin(grey), nebulin (light blue), a-actinin (dark red), desmin (green) and myotilin (red). A1: twonormal sarcomeres are shown; A2: a-actinin is lost, and F-actin and myotilin are increased in the middle Z-disc. A3: The Z-disc is broadened: F-actin and myotilin are increased and a-actinin is present at the edges of the broadened Z disc. A4: A new sarcomere is formed with insertion of myosin, titin and nebulin. Note that desmin in the areas of remodelling is increased and also present as longitudinal strands. B1: A normal sarcomere is shown. B2: The sarcomere is broadened. F-actin, myotilin and desmin are increased, and a-actinin, myosin, titin and nebulin are lost. B3: a Actinin is reintegrated in the middle of the broadened sarcomere forming a new Z-disc. Subsequent addition of the other myofibrillar proteins is not coordinated in the two sarcomeres. B4: An additional sarcomere has been added to the myofibril. Contact Thornell Lars-Eric and Lena Carlsson Umeå University Department of Integrative Medical Biology Section for Anatomy Email: lars-eric.thornell@anatomy.umu.se icsspe.org/index.php?m=15 New Aspects on Myofibrillar Remodeling in Human Muscle after Eccentric Exercise Lars-Eric Thornell & Lena Carlsson