Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury - PubMed (original) (raw)

Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury

Richard M Lovering et al. Am J Physiol Cell Physiol. 2004 Feb.

Abstract

The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male Sprague-Dawley rats at 900 degrees /s throughout a 90 degrees -arc of motion. Maximal tetanic tension (Po) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (alpha-actinin, desmin, dystrophin, beta-spectrin), integral membrane (alpha- and beta-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 was significantly reduced to 4.23 +/- 0.22 N, compared with 8.24 +/- 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 +/- 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (beta-spectrin) or not at all (alpha- and beta-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction.

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Figures

Fig. 1

Fig. 1

Membrane damage with loss of function. A: integrity of the sarcolemma was assessed by permeation of Evans blue dye (EBD). Noninjured TAs did not show dye uptake into the fibers (left), whereas injured TAs did (right), indicating sarcolemma damage. B: the percentage of EBD-positive fibers (dashed line) is plotted against contractile function (solid line), as measured by maximal isometric tetanic tension (Po). EBD was found in a high percentage of fibers immediately after the injury, when Po dropped significantly from noninjured values. Minimal EBD was seen 21 days postinjury, when contractile function was fully restored.

Fig. 2

Fig. 2

Labeling of membrane-associated cytoskeleton after injury. Tissue sections from injured tibialis anterior muscles (TAs) were labeled for laminin (A–C), α-dystroglycan (D–F), and β-dystroglycan (G–I). The left column shows images of the respective molecules of interest. EBD-positive fibers from the same section are shown in the middle column, and the right column shows the overlay of the EDB (red) with fluorescent labeling (green). EBD was administered by intraperitoneal injection 24 h before injury. The data indicate that the organization of membrane-associated molecules is not affected. Scale bar = 50 μm.

Fig. 3

Fig. 3

Labeling of the subsarcolemmal cytoskeleton after injury. Tissue sections from injured TAs were labeled for dystrophin (AC), β-spectrin (D–F), and desmin (G–I). The left column shows images of the respective molecules of interest. EBD-positive fibers from the same section are shown in the middle column, and the right column shows the overlay of the EDB (red) with fluorescent labeling (green). EBD was administered by intraperitoneal injection 24 h before injury. Fibers that were permeable to EBD also lacked dystrophin (arrows, A–C). β-Spectrin and desmin were much less affected in injured fibers (arrows, D–F and G–I, respectively). Scale bar = 50 μm.

Fig. 4

Fig. 4

Double labeling of dystrophin and desmin. Fluorescent confocal images (×25) from double labeling of dystrophin and desmin of uninjured (A–C) and injured (D–F) tissue sections. Sections were labeled with monoclonal antibodies against dystrophin (B and E and light blue in overlay C or yellow/green in overlay F) and polyclonal antibodies against desmin (A and D and blue in C and F). In noninjured muscle, desmin labeling is throughout the fibers and dystrophin labeling is circumferential as expected. In injured fibers, loss of desmin labeling is more diffuse (asterisk in D), and there is incomplete labeling of dystrophin (arrow in E). Scale bar = 25 μm.

Fig. 5

Fig. 5

Loss of dystrophin labeling is confined to the COOH terminus. Serial sections from an injured TA are labeled with monoclonal antibodies to either the rod domain (dys1, A) or the COOH terminus (dys2, B), showing a selective loss of labeling to the COOH terminus. Scale bar = 50 μm.

Fig. 6

Fig. 6

Western blot analysis of dystrophin and desmin from injured TA muscles. Western blot analysis was performed on injured TA at various time points after injury. NI are noninjured TAs, and D0 represents TAs harvested within 15 min postinjury. Data are also included for 3, 7, and 21 days after injury (D3, D7, D21, respectively). Each lane contains 20 μg of protein. A significant drop in the chemiluminescent signal is seen for dystrophin after injury (A). Although modulated, desmin does not show the same loss (B). The nonmembrane protein α-actinin was used as a control (C). Each bar represents data from 3 independent experiments, and the histograms represent means ± SD.

Fig. 7

Fig. 7

Schematic of selected membrane-associated molecules.

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