Dysferlin overexpression in skeletal muscle produces a progressive myopathy

Abstract

Objective: The dose-response effects of dysferlin transgenesis were analyzed to determine if the dysferlin-deficient myopathies are good candidates for gene replacement therapy.

Methods: We have generated 3 lines of transgenic mice, expressing low, mid, and high levels of full-length human dysferlin from a muscle-specific promoter. Transgenic skeletal muscle was analyzed and scored for morphological and functional deficits.

Results: Overexpression of dysferlin in mice resulted in a striking phenotype of kyphosis, irregular gait, and reduced muscle mass and strength. Moreover, protein dosage correlated with phenotype severity. In contrast to dysferlin-null skeletal muscle, no evidence of sarcolemmal impairment was revealed. Rather, increased levels of Ca(2+)-regulated, dysferlin-binding proteins and endoplasmic reticulum stress chaperone proteins were observed in muscle lysates from transgenic mice as compared with controls.

Interpretation: Expression levels of dysferlin are important for appropriate function without deleterious or cytotoxic effects. As a corollary, we propose that future endeavors in gene replacement for correction of dysferlinopathy should be tailored to take account of this.

Figure 1. Generation of transgenic mice overexpressing dysferlin

a, Schematic diagram of hDysf transgene used to generate dysferlin transgenic mice. The human skeletal actin promoter (2.2kb) drives skeletal muscle-specific expression. The hybrid HSA/vp1 intron is situated between the promoter and cDNA, containing splice donor and acceptor sequences. The SV40 poly(A) signal follows the dysferlin cassette. b, RT-PCR analysis of hDysf transgene (HSA-hDysf), total dysferlin including mouse endogenous (mDysf/hDysf) and ribosomal gene RPS7 expression levels in skeletal muscle from wild-type (WT) mice and three transgenic lines overexpressing hDysf at different levels: hDysf-low, hDysf-mid and hDysf-high. c, Upper panel, Western blot analysis of human dysferlin protein expression in hindlimb skeletal muscle lysates from wild-type mice, and three hDysf transgenic lines. Immunoblotting was performed with monoclonal antibodies that recognize either human-specific dysferlin alone (Dysf (H)) or human and mouse dysferlin (Dysf (M; H)). Actin was monitored as a loading control. Lower panel, Protein lysates were prepared from hDysf-high transgenic mice tissues and immunoprobed with human-specific dysferlin antibody. Exogenous, transgenic Dysf expression is observed only in cardiac and skeletal muscle lysates. d, Immunoblot analysis of dysferlin expression (endogenous and transgenic) in muscle lysates from wild-type and hDysf mice. e, Subcellular fractionation of microsomal homogenates from wild-type and hDysf-low mice reveal that transgenic dysferlin is enriched in sarcoplasmic reticular fractions as well as in the sarcolemma. SL, sarcolemma; LSR, light sarcoplasmic reticulum; TRI, triads; HSR, heavy sarcoplasmic reticulum; P, pellet.

Figure 2. Increased transgenic dysferlin overexpression correlates with muscle atrophy and reduced body mass

a, Body weight gain of wild-type (WT) and hDysf transgenic mice (n=5 per group) was monitored biweekly from 4-16 weeks of age. Values are means ± SD. Transgenic animals displayed consistently reduced body weight compared to wild-type controls (group hDysf low (P < 0.05) and groups hDysf-mid and -high (P < 0.01 by ANOVA)). b, Ratio of food consumption to body mass in WT and hDysf transgenic mice (n=3 mice per group). Values are means ± SD. c, Photographs of wild-type control and hDysf over-expressing transgenic mice at 8 weeks of age. hDysf-mid and –high level transgenic mice are kyphotic with an irregular gait and display atrophy of the limb musculature. d, Relative muscle weights. The weights of five muscle groups (n=4 mice per group) from hDysf transgenic mice were measured and normalized to weights of corresponding muscles in age-matched littermate wild-type controls. Values are means ± standard error (*P < 0.05; **P <0.001, t-test).

Figure 3. Hindlimb strength and single muscle fiber contractile properties in hDysf-transgenic mice

a, Hindlimb grip strength of WT and hDysf-transgenic mice (n=3 per group) was measured from 10 to 25 weeks of age and grip strength in newtons (N) was normalized to body weight (kg). Each bar represents the mean ± standard error of values measured. hDysf-mid and hDysf-high transgenic mice displayed attenuated hindlimb strength compared to wild-type controls (P < 0.05 and P < 0.01, respectively, by ANOVA) b, The slack test was performed on isolated single myofibers from hDysf-mid transgenic and wild-type mice from quadriceps (WT, n=14 fibers; hDysf Tg, n=10 fibers) and gastrocnemius muscle (WT, n=18 fibers; hDysf Tg, n=10 fibers). Data are presented for Type IIb fibers only because other fiber types were not present in both the wild type and transgenic mice. Maximal force (Po) and maximum unloaded shortening velocity (Vo) was measured. Values are means ± SD (*P < 0.05).

Figure 4. Immunofluorescence and western blot analyses of sarcolemmal and dysferlin-binding proteins in hDysf transgenic muscle

a, Immunofluorescence staining of quadriceps muscle cross-sections from wild-type (WT) and hDysf-mid transgenic mice with human-specific (Dysf (h)) and mouse/human specific antibodies to dysferlin (Dysf (m/h)). Dysferlin immunostaining is both sarcolemmal and intracellular, visible as punctate vesicular, perinuclear (arrowhead) and cytoplasmic aggregate staining (arrows). b, Normal immunofluorescnce staining for caveolin-3 (Cav-3) and DGC components β-dystroglycan (β-DYS) and dystrophin (DYS). Scale bar, 100μm. c, Upper panel Immunoblot analysis of quadriceps microsomal lysates from from wild-type (WT) and hDysf-mid transgenic mice (n=3 per group), using antibodies to annexin A1 (AnxA1), annexin A2 (AnxA2), caveolin-3 (Cav-3), calpain-3, β-dystroglycan (β-DYS); β-tubulin staining was monitored as a loading control. Lower panel, Densitometric analysis of band intensity normalized to β-tubulin, expressed as fold change over wild-type (WT) levels. Values are means ± standard error (*P < 0.05; **P < 0.01; ***P <0.001, unpaired t-test).

Figure 5. Expression of ER stress proteins in hDysf transgenic muscle

a, Representative NADH-tetrazolium reductase histochemical staining of hDysf-mid transgenic quadriceps muscle reveals tubular aggregate-like structures (arrows) and intra-sarcoplasmic vacuoles (arrowheads). b, Western blot analysis of whole cell protein lysates from wild-type (WT) and hDysf-mid transgenic mice (n=3 per group), using antibodies to the endoplasmic reticulum stress proteins glucose-related protein 78 (GRP78), GADD150/CHOP and calreticulin; α-actin used as a loading control. Lower panel, Densitometric analysis of band intensity normalized to α-actin, expressed as fold change over wild-type (WT) levels. Values are means ± standard error (*P < 0.05; ***P <0.001, unpaired t-test).