Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.
Authors
Karen K. McKee, Stephanie C. Crosson, Sarina Meinen, Judith R. Reinhard, Markus A. Rüegg, Peter D. Yurchenco
(A–C) H&E staining of cross sections of the corresponding region of 11-week-old extensor carpi radialis muscle. (D–F) Picrosirius red collagen staining of adjacent sections. White arrows indicate examples of central nuclei in magnified insets (original magnification, ×3). The morphology of rounded, variable-sized myofibers with increased interfiber collagen–positive staining, characteristic of dystrophic muscle, was improved by the transgene to more closely resemble control muscle morphology. Scale bars: 100 μm. (G) Histogram showing the distribution of cross-sectional myofiber areas comparing control, dy2J, and Tg+dy2J mice (n = 5 mice/condition; data represent the average ± SD). Inset graph in G shows the overall myofiber cross-sectional area (average ± SEM, n = 5/condition). Histogram plot shows a smaller size distribution for dy2J muscle but a normal distribution and overall average for Tg+dy2J muscle. (H) Average (mean ± SD, n = 5 mice/condition) of the sum of myofiber cross-sectional areas. The area reduction in dy2J muscle was corrected by the transgene. (I) Number of myofibers per muscle (average ± SD, n = 5/condition). The number of myofibers was reduced in dy2J muscle and increased to levels indistinguishable from those of control muscle with the transgene. (J) The fraction of myofibers (average ± SD, n = 5/condition) with central nuclei (regeneration marker), elevated in dy2J muscle, was decreased in the presence of the transgene. (K) Picrosirius red fluorescence, a collagen stain, was reduced to levels indistinguishable from those in control muscle (average ± SD, n = 5/condition). Individual mouse values are superimposed on the average bars in panels G–K. P value determined in G–K by 1-way ANOVA followed by pairwise Holm-Sidak test.