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) αLNNd is a chimeric protein consisting of murine Lmα1 LN-LEa domains fused to the C-terminal moiety of nidogen-1. Its corresponding cDNA was inserted into a vector driven by the MCK promoter and used to generate transgenic mice. (B) Muscle Lm211 and its homolog Lm111 bind to nidogen-1 (Nd), agrin, αDG, and α7β1 integrin. Polymerization results from the binding of the LN domains (PD) of the Lmα2 (or α1), Lmβ1, and Lmγ1 subunits to form a ternary node at the N-termini of each laminin. The α2LN domain is defective in the dy2J mouse, greatly reducing self-assembly. αLNNd can compete with and replace native nidogen-1, binding to the Lmγ1-LEb3 domain locus, to create a new short arm capable of binding to the other LN domains. (C) αLNNd protein expression was probed by immunoblotting of extracts from 3- and 8-week-old triceps (Tr) and tibialis anterior (TA) (goat anti–Lmα1-LN Ab). Expression was detected in αLNNd-Tg but not WT muscle. (D) Immunostained sections of triceps muscle from 3-week-old WT and αLNNd-Tg mice revealed a sarcolemmal BM distribution of αLNNd (anti-F2, reacting with Lmα1LN-LEa) similar to that of Lmα2 (rat anti-Lmα2). Scale bar: 100 μm.