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.
Karen K. McKee, Stephanie C. Crosson, Sarina Meinen, Judith R. Reinhard, Markus A. Rüegg, Peter D. Yurchenco
(A) Combined extracts (B plus C plus E) from 4-week-old forelimb dy2J and Tg+dy2J muscle were immunoprecipitated twice with αLNNd-specific Ab (anti-F2, IP #1 and IP #2). The αLNNd-depleted supernatants were then immunoprecipitated with anti-nidogen G2-G3 (IP #3 and IP #4). Precipitates, following SDS-PAGE and membrane transfer, were immunoblotted with anti-Lmβ1γ1 (upper cut membrane, dotted cut line shown) and anti-nidogen G2-G3 (lower cut membrane). αLNNd was detected only in the Tg+dy2J muscle. (While a small amount of nidogen was present in the anti-F2 immunoprecipitates of the dy2J control muscle, this band was similarly seen in immunoblots precipitated with anti-IgY agarose beads alone and deduced to result from nonspecific nidogen binding to beads). Following αLNNd depletion, isolated nidogen was immunoprecipitated with anti–nidogen G2-G3 Ab. The upper αLNNd band was distinguished from the adjacent nidogen band to determine the relative amounts in the lysates (IP #1 plus IP #2). To estimate the fraction of β1γ1-laminins bound to αLNNd and to nidogen-1, the laminin band intensities were multiplied by the fraction of αLNNd and nidogen-1 in each blot to determine the distribution and then summed. (B) Plot (average ± SD, n = 4 mice/condition, P = 0.007, by 2-tailed t test) of the calculated fraction of αLNNd relative to the total of αLNNd plus nidogen and the fraction of β1γ1 laminins bound to αLNNd relative to total β1γ1 laminins. Lm, laminins.