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–K) Sandwich ELISA determination of laminin content in forelimb muscle. ELISA plates coated with rabbit polyclonal anti-Lmα2 (A, B, E, and F), anti-Lmα4 (C and D), anti-Lmβ1γ1 (G and H), or anti-nidogen G2-G3 domains (I and J) were incubated with serial dilutions of total combined lysate fractions (A–D) or “matrix” (collagenase plus EDTA plus SDS) fractions (E–J) extracted from muscle from four 4-week-old control (+/+ and dy2J/+), dy2J-mutant, and αLNNd-Tg dy2J/dy2J mice. Laminins were detected by ELISA after treatment with Lmβ1γ1-specific Ab (A–F, I, and J) or Lmα4-specific Ab (G and H). Plots show the relative average laminin absorbance (± SD, n = 4/condition) for Lm211 (total, B), Lm411 (total, D), Lm211 (matrix, F), Lm411 (matrix, H), and total nidogen-bound laminins (matrix, J). (K) GAPDH and α-actinin blots for each mouse muscle shown, with the latter used as a myofiber denominator. Lm211 was reduced, while Lm411 was increased in dy2J muscle lysates compared with control muscle total lysates, with no significant change in transgenic mouse muscle lysates. However, the pooled matrix fractions, after removal of soluble and loosely bound protein, contained significant increases of Lm211 and nidogen-binding laminins in Tg+dy2J muscle compared with dy2J muscle. To relate laminin levels to muscle α-actinin, the averaged A450/lysate volume ratios in A, C, E, G, and I were divided by the relative α-actinin immunoblot (K) intensities (A/α-act), shown in the corresponding plots in B, D, F, H, and J. (L–R) Changes in relative muscle mRNA by qRT-PCR (ΔΔCt) at 4 weeks revealed that Lama2, Lamc1, and dystroglycan levels were not significantly different in WT, dy2J, or Tg+dy2J muscle. Lmα4 (Lama4) levels were similarly increased in dy2J and Tg+dy2J muscle (but not increased in Tg+dy2J muscle relative to dy2J muscle alone), while integrins α7 and β1 and fibronectin (inflammatory marker) were increased in dy2J muscle and reduced in Tg+dy2J muscle. The presence of αLNNd did not change the Lama4 mRNA levels in WT mice. Average and SD plots are shown (n = 4 mice/condition in L–P and R; n = 3 for control and n = 4 for dy2J and Tg+dy2J mice in Q). Graphs also show individual mouse values (colored circles) superimposed on bars. Statistical significance was determined by 1-way ANOVA with Holm-Sidak comparisons. Av, average.