Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype
Karen K. McKee, … , Markus A. Rüegg, Peter D. Yurchenco
Karen K. McKee, … , Markus A. Rüegg, Peter D. Yurchenco
Published February 20, 2017
Citation Information: J Clin Invest. 2017;127(3):1075-1089. https://doi.org/10.1172/JCI90854.
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Research Article Muscle biology

Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype

Abstract

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

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Figure 7

Effect of αLNNd on nonpolymerizing laminin assembly on C2C12 myotubes.

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Effect of αLNNd on nonpolymerizing laminin assembly on C2C12 myotubes. (...
(A) Lm111, in which the α1-short arm has been deleted (LmαΔLN-L4b), binds to αLNNd. (B–E) Lawns of fused C2C12 myotubes were incubated with the indicated WT and polymerization-deficient Lm111 (28 nM) in the presence of nidogen-1 (28 nM) and collagen IV (14 nm), without or with αLNNd (28 nM) for 1 hour, followed by washing, fixation, and immunostaining. Representative myotube images are shown in B (Lmγ1 immunofluorescence) and D (collagen IV immunofluorescence) (scale bar: 200 μm), with corresponding graphs of the average summed intensities ± SD (n = 7–9 × 10 fields/condition; 1-way ANOVA with pairwise Holm-Sidak comparisons) shown in C and E. A substantial decrease of nonpolymerizing LmαΔLN-L4b accumulation compared with WT control was prevented when αLNNd was coincubated with LmαΔLN-L4b. In contrast, αLNNd did not alter WT laminin accumulation. Smaller collagen IV differences were detected among polymerizing and nonpolymerizing laminins. IF, immunofluorescence; Lm, laminin.