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. 2005 Jan;166(1):253-63.
doi: 10.1016/s0002-9440(10)62249-3.

Transgenic Expression of {alpha}7{beta}1 Integrin Maintains Muscle Integrity, Increases Regenerative Capacity, Promotes Hypertrophy, and Reduces Cardiomyopathy in Dystrophic Mice

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Transgenic Expression of {alpha}7{beta}1 Integrin Maintains Muscle Integrity, Increases Regenerative Capacity, Promotes Hypertrophy, and Reduces Cardiomyopathy in Dystrophic Mice

Dean J Burkin et al. Am J Pathol. .
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Abstract

We previously reported that enhanced expression of the alpha7beta1 integrin ameliorates the development of muscular dystrophy and extends longevity in alpha7BX2-mdx/utr(-/-) transgenic mice (Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ: Enhanced expression of the alpha7beta1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. We now report on the mechanism by which these mice were rescued by the integrin. As a result of increased integrin in alpha7BX2-mdx/utr(-/-) mice the structural integrity of the myotendinous and neuromuscular junctions are maintained. A twofold increase in satellite cells in alpha7BX2-mdx/utr(-/-) skeletal muscle was detected by immunofluorescence using the satellite cell marker c-met. These cells enhanced the regenerative capacity of muscle in the transgenic animals as determined by fusion of BrdUrd-labeled cells into muscle fibers. Increased integrin also leads to hypertrophy. Finally, transgenic expression of alpha7BX2 integrin chain in skeletal muscle secondarily reduces the development of cardiomyopathy, the ultimate cause of death in these animals. We believe this multiplicity of responses to increased alpha7beta1 integrin collectively inhibits the development of muscle disease and increases longevity in these mice.

Figures

Figure 1
Figure 1
Dystrophin-associated proteins remain unchanged in α7BX2 transgenic mice. Western blot analysis of DAPs isolated from wild-type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice. An absence of dystrophin and reduction in β-dystroglycan, dystrobrevin, and sarcospan in 5- and 10-week-old mdx and mdx/utr−/− mice were observed as previously reported. Likewise, α7BX2 transgenic mice show a similar absence or reduction in these DAPs. Samples from two mice per genotype are shown and at least six mice per genotype were analyzed.
Figure 2
Figure 2
Myotendinous and neuromuscular junctions are maintained in α7BX2 transgenic mice. A: Structure of the myotendinous junctions of gastrocnemius muscle of 5-week-old wild-type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice. The sarcolemma of the myotendinous junction in wild-type and mdx mice is highly folded (arrows), increasing the contact between muscle and tendon. In contrast, the sarcolemma of the myotendinous junction in the severely dystrophic mdx/utr−/− mice exhibits little folding. Enhanced expression of the α7BX2 transgene in mdx/utr−/− mice results in maintenance of sarcolemmal folding similar to that seen in wild-type and mdx mice. B: Quantitation of sarcolemmal folds at the myotendinous junction reveals a slight reduction in folding in mdx mice compared to wild-type. In contrast, a sixfold reduction in sarcolemmal folds at the myotendinous junction, was scored in mdx/utr−/− mice. Expression of the α7BX2 transgene maintains near normal folding of the sarcolemma in mdx/utr−/− mice. C: A mild reduction in postsynaptic folding of the neuromuscular junctions of sternomastoid muscles is observed in mdx mice compared to wild-type animals whereas a major reduction in junctional folds occurs in mdx/utr−/− mice. Expression of the α7BX2 transgene in mdx/utr−/− mice results in a maintenance of postsynaptic folding comparable to that observed in mdx mice. Folds at the myotendinous and neuromuscular junctions were counted for at least 10 junctions per animal (n = 3 mice per genotype). The standard errors of the mean values for each animal within each group are indicated. P < 0.05 for comparisons of transgenic and nontransgenic mice.
Figure 3
Figure 3
Increased satellite cell number in the muscle of α7BX2 transgenic mice. A: Antibody against the satellite cell marker c-met (hepatocyte growth factor) was used to identify these cells in sections of the lower hindlimb from 5-week-old wild-type, mdx, mdx/utr−/−, and α7BX2-mdx/utr−/− mice (green, arrow). C-met-positive cells in 50 fields were scored. Mean numbers (±SEM) for at least six animals of each genotype are given. Wheat germ agglutinin staining delineates fibers (red). B: A twofold increase in satellite cells was detected in hindlimb muscle of α7BX2-mdx/utr−/− transgenic mice compared to nontransgenic animals (P < 0.05).
Figure 4
Figure 4
Increased integrin promotes repair. To determine whether the satellite cells were used in repair, mice were injected with BrdUrd to label replicating cells. Seventy-two hours later muscle sections were analyzed by immunofluorescence for BrdUrd incorporation into DNA (green). Wheat germ agglutinin staining delineates fibers (red). Nuclei are stained with DAPI (blue). BrdUrd-labeled central nuclei (arrows) in 50 random fields were scored for each animal. Mean numbers (±SEM) are given for 11 animals for each genotype. For mdx/utr−/− mice, the value is 32.0 ± 4.56 and for α7BX2-mdx/utr−/− 55.0 ± 7.35 (P = 0.0152, unpaired t-test). Thus increased integrin expands the reservoir of satellite cells and the regenerative capacity of dystrophic muscle.
Figure 5
Figure 5
Increased expression of α7β1 integrin promotes hypertrophy. Cross-sectional areas of fibers in soleus and tibialis anterior muscles were measured. Using the largest fiber area in wild-type animals as a cutoff point (dashed line), α7BX2-mdx/utr−/− mice were found to have four times more hypertrophic fibers than mdx/utr−/− mice. Thus increasing the level of integrin promotes a hypertrophic response that contributes to the rescue of these animals. The cross-sectional areas of 200 to 300 fibers from each muscle were measured for each of four animals per genotype.
Figure 6
Figure 6
α7BX2-mdx/utr−/− mice exhibit reduced cardiomyopathy. A: α7BX2-mdx/utr−/− mice have a fourfold reduction in the area of fibrotic lesions compared to mdx/utr−/− mice as evidenced by H&E staining of 8-week cardiac muscle (dashed outline). No lesions were observed in wild-type or mdx mice. B: Similarly, transgenic α7BX2-mdx/utr−/− mice showed a 10-fold reduction in the area of Evans blue dye uptake compared to mdx/utr−/−mice (arrows) and no dye uptake was observed in wild-type or mdx mice. C: Ventricular expression of ANF is an indicator of cardiomyopathy. RT-PCR analysis of ANF RNA shows that it is highly elevated in 8-week ventricular muscle of mdx/utr−/− mice and comparatively it is markedly reduced in α7BX2-mdx/utr−/− mice (P < 0.0002). All values are normalized to GAPDH.

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