Molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a truncated dystrophin

Abstract

Myotendinous strain injury is the most common injury of human skeletal muscles because the majority of muscle forces are transmitted through this region. Although the immediate response to strain injury is well characterized, the chronic response to myotendinous strain injury is less clear. Here we examined the molecular and cellular adaptations to chronic myotendinous strain injury in mdx mice expressing a microdystrophin transgene (microdystrophin(DeltaR4-R23)). We found that muscles with myotendinous strain injury had an increased expression of utrophin and alpha7-integrin together with the dramatic restructuring of peripheral myofibrils into concentric rings. The sarcolemma of the microdystrophin(DeltaR4-R23)/mdx gastrocnemius muscles was highly protected from experimental lengthening contractions, better than wild-type muscles. We also found a positive correlation between myotendinous strain injury and ringed fibers in the HSA(LR) (human skeletal actin, long repeat) mouse model of myotonic dystrophy. We suggest that changes in protein expression and the formation of rings are adaptations to myotendinous strain injury that help to prevent muscle necrosis and retain the function of necessary muscles during injury, ageing and disease.

Figure 1.

Chronic myotendinous strain injury at the Achilles myotendinous junction (MTJ) in mdx mice expressing the microdystrophin^ΔR4–R23 transgene. (A) The molecular structure of truncated dystrophins. Dystrophin contains two actin-binding domains (ABD), a large central rod domain, a cysteine-rich region (CR) and a C-terminal (CT) domain. The central rod domain is composed of four hinge regions and 24 spectrin-like repeats. The transgenes used in this study are shown below the full-length dystrophin. Mdx mice expressing microdystrophin^ΔR4–R23 and minidysGFP (ΔH2-R19/ΔCT, GFP) transgenes have been published previously (24,25). MinidysGFP has an enhanced green fluorescence protein (GFP) in place of the CT domain. (B) Dystrophin, microdystrophin^ΔR4–R23 and minidysGFP (all shown in green) concentrate in the MTJ in the gastrocnemius muscle with laminin (shown in red). Nuclei are stained with 4′,6-diamidino-2-phenylindole in blue. Toluidine blue images show that tears in the MTJ can associate with sites of myofibril degeneration. White arrows show sites of failure within the MTJ (B) and tendon (C) in microdystrophin^ΔR4–R23/mdx mice. Black arrows show the accompanying pathology. (D) Electron microscopy images show a rippled appearance of the MTJs, thickened Z-lines and absence of I-bands in microdystrophin^ΔR4–R23/mdx transgenic mice. (E) The mean ± SE length of folds at the Achilles MTJ. Note that the length of folds are reduced in mdx mice and restored by the truncated dystrophin transgenes. ***P < 0.001 compared with wild-type. Scale bar = 2 µm for electron microscopy images and 10 µm for other images.

Figure 2.

Expression and localization of α-dystrobrevin, α7-integrin and utrophin in wild-type, mdx, microdystrophin^ΔR4–R23/mdx and minidysGFP/mdx mice. (A) Note that the truncated dystrophins restored α-dystrobrevin to the myotendinous junction (MTJ) in mdx mice. α7-integrin and utrophin were qualitatively increased in mdx and microdystrophin^ΔR4–R23/mdx MTJs, but not in minidysGFP/mdx mice. (B) Expression of cytoskeletal proteins within the gastrocnemius and diaphragm muscles. All lanes were loaded equally as represented by α-actin. Note that α7-integrin and utrophin were increased in microdystrophin^ΔR4–R23/mdx gastrocnemius muscles with myotendinous strain injury, but not in the diaphragm muscles that have no signs of myotendinous strain injury.

Figure 3.

Ringed fibers in mdx mice expressing the microdystrophin^ΔR4–R23 transgene. (A) Electron micrographs of two adjacent gastrocnemius muscle fibers from wild-type, mdx, microdystrophin^ΔR4–R23/mdx and minidysGFP/mdx mice. Scale bars = 2 µm. (B) Ringed fibers in myofibers from microdystrophin^ΔR4–R23/mdx gastrocnemius muscles stained with toluidine blue. Scale bar = 50 µm. (C) Transverse sections of muscle immunolabeled for desmin (green) and dystrophin (red). Scale bar = 50 µm. (D) Mean ± SD percentage of ringed fibers in various microdystrophin^ΔR4–R23/mdx muscles. Scale bar = 50 µm. Arrows point to examples of rings. ***P < 0.001 and **P < 0.01 compared with diaphragm; ^###P < 0.001 compared with gastrocnemius and quadriceps.

Figure 4.

Gastrocnemius muscles expressing the microdystrophin^ΔR4–R23 transgene were significantly protected from contraction-induced injury. (A) The contractile performance of gastrocnemius muscles immediately prior to increasing length changes during maximal force production for wild-type, mdx, microdystrophin^ΔR4–R23/mdx transgenic and minidysGFP/mdx transgenic mice. Points represent the mean ± SD percentage of the initial optimal muscle contraction. Microdystrophin^ΔR4–R23/mdx was significantly increased compared with wild-type (*P < 0.05; **P < 0.01; ***P < 0.001). mdx was significantly reduced compared with wild-type (^#P < 0.05; ^##P < 0.01). (B) Gastrocnemius muscles after intravenous administration of Evans blue dye (EBD), before and after 33% stretch. EBD enters muscle fibers that have holes in the sarcolemma. Arrows point to EBD in fibers near the ends of the wild-type muscles after 33% stretch. (C) Cross-section of wild-type muscle showing no EBD without stretch-induced injury. Nuclei are shown in blue. (D) Cross-section of wild-type muscle stretched 33% beyond its optimal length. Note that many wild-type muscle fibers have EBD (in red) showing that contraction-induced injury tears the sarcolemma. (E) Representative longitudinal section of microdystrophin^ΔR4–R23/mdx muscle that has not been stretched. Arrow points to EBD at the MTJs (myotendinous junctions). EBD is also present in a single muscle fiber. (F) Cross-section of microdystrophin^ΔR4–R23/mdx muscle stretched 33% beyond its optimal length. Note the lack of EBD showing the sarcolemma was significantly protected from contraction-induced injury in the eccentric stretch assay. Scale Bar = 50 µm.

Figure 5.

Correlation between myotendinous strain injury and ringed fibers in the HSA^LR (human skeletal actin, long repeat) mouse model of DM (myotonic dystrophy). (A) Longitudinal resin section of the Achilles MTJ (myotendinous junction) stained with toluidine blue. Arrow points to a partial tear. Arrowheads point to accompanying pathology and infiltration of adipocytes. Scale bar = 5 µm. (B) Transverse section of the gastrocnemius stained with toluidine blue. Scale bar = 20 µm. (C) Electron microscopy image of a ringed fiber. Scale bar = 2 µm. Arrows in (B) and (C) point to the rings.

Figure 6.

Model of acute and chronic myotendinous strain injury. Details are described in Discussion.