Myocilin interacts with syntrophins and is member of dystrophin-associated protein complex

Abstract

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

FIGURE 1.

Myocilin expression in differentiating C2C12 cells. A, Western blot analysis of myocilin expression in undifferentiated myoblasts (day 0) and differentiating myotubes (day 5). Equal amounts of lysates were probed with antibodies against myocilin (1:4000 dilution) and HSC70 (1:2000 dilution). B, relative abundance of Myoc mRNA in mouse myoblasts (day 0) and myotubes (day 5) (n = 3 each) was evaluated on the basis of Ct values obtained by real-time PCR. Normalization was performed using GAPDH mRNA. Error bars, S.E. **, p < 0.01. C, myoblasts (day 0) and myotubes (day 5) were stained with antibodies against myosin heavy chain (MHC; 1:100 dilution) and myocilin (1:100 dilution) as described under “Experimental Procedures.” Scale bar, 100 μm. D, myocilin is not efficiently secreted from myotubes. C2C12 myoblasts were differentiated for 5 days and maintained in serum-free medium for an additional 5 days. The lysate and conditioned medium were probed with anti-myocilin antibodies (1:4000 dilution) and anti-pigment epithelium-derived factor antibodies (PEDF; 1:5000 dilution).

FIGURE 2.

Interaction of myocilin and α1-syntrophin in yeast and mammalian cells. A, yeasts co-expressing α1-Syn and the indicated myocilin fusion proteins were spotted on −LT medium to confirm mating and on −AHLT medium to confirm protein-protein interactions between bait and prey proteins. B and C, lysates of C2C12 myotubes (B) or skeletal muscles (C) were immunoprecipitated with antibodies against myocilin or pan-syntrophin. Immunoprecipitates were collected, separated by SDS-PAGE, and then probed with HRP-conjugated pan-syntrophin antibodies or myocilin antibodies. Rabbit or mouse IgG was used as a control. Input lanes contained 5% of lysates used for immunoprecipitation. aa, amino acids; IP, immunoprecipitation; IB, immunoblot.

FIGURE 3.

Co-localization of myocilin and α1-syntrophin at the membrane of C2C12 myotubes. C2C12 myotubes were stained with antibodies against α1-Syn and myocilin (top row) or myosin heavy chain (MHC) and myocilin (bottom row). The inset in the bottom right corner of each image shows the higher magnification for the area surrounded by the dotted line. The yellow color in merged panels represents co-localization. Scale bar, 20 μm; scale bar in inset, 10 μm.

FIGURE 4.

Overexpression of myocilin in vivo induces relocalization of α1-syntrophin. A, total muscle lysates were prepared from 8-month-old wild-type and transgenic (Tg) mice. Equal amounts of muscle lysates were probed with antibodies against myocilin (1:4000 dilution), α1-Syn (1:2000 dilution), and HSC70 (1:2000 dilution). B, myocilin immunofluorescence was performed using 10 μm frozen sections of the TA muscles from 8-month-old wild-type and transgenic mice. C, frozen sections of TA muscle from transgenic mice was double-stained with antibodies against myocilin and α1-Syn. The merged green and red signals indicate their co-localization at the sarcolemma. D, α1-Syn immunofluorescence was performed on frozen cross-sections as in B. Higher magnification of the areas surrounded by dotted lines is shown to the right. Scale bar, 100 μm; scale bar in right-hand raw images in D, 20 μm.

FIGURE 5.

Overexpression of myocilin in vivo induces relocalization of DAPC components to dystrophin and the binding of α-DG to laminin. A, total muscle lysates prepared from 8-month-old wild-type and transgenic (Tg) mice were probed with antibodies against dystrophin (1:20 dilution), α-dystrobrevin (1:1000 dilution), β-DG (1:1000 dilution), nNOS (1:1000 dilution), and HSC70 (1:2000 dilution). Density ratios were calculated from two independent experiments using the ImageJ program. B, the indicated muscle lysates were immunoprecipitated with antibodies against dystrophin. Immunoprecipitates were probed with antibodies against dystrophin (1:20 dilution), myocilin (1:4000 dilution), α1-Syn (1:4000 dilution), and nNOS (1:1000 dilution). C, WGA elutes from digitonin-solubilized muscle lysates were probed with antibodies against dystrophin (1:20 dilution) and β-DG (1:1000 dilution). D, quantification of the results shown in C. Levels of dystrophin were normalized relative to β-DG levels from three independent experiments using the ImageJ program. Error bars, S.E. **, p < 0.01. E, WGA elutes were probed with α-DG (IIH6; 1:500 dilution) and further analyzed by a laminin overlay assay.

FIGURE 6.

Changes in muscle hypertrophy and atrophy signaling pathways in myocilin expressing transgenic mouse. A, total lysates from C2C12 myotubes differentiated on the plates with or without laminin were probed with antibodies against phospho-AKT1 (P-AKT1; 1:1000 dilution) and total AKT1 (1:2000 dilution). B, total muscle lysates were prepared from 8-month-old wild-type and transgenic (Tg) mice. Equivalent amounts of muscle lysates were separated by SDS-PAGE and then probed with antibodies against phospho-AKT1 (1:1000 dilution), total AKT1 (1:2000 dilution), phospho-FoxO3a (1:1000 dilution), FoxO3a (1:1000 dilution), and HSC70 (1:2000 dilution). C, mRNA levels of MuRF-1 and atrogin-1 in the muscles of 8-month-old wild-type and transgenic mice (n = 3) were quantified by real-time PCR. Error bars, S.E. *, p < 0.05; **, p < 0.01. D, total muscle lysates as in B were probed with antibodies against phospho-mTOR (P-mTOR; 1:500 dilution), total mTOR (1:500 dilution), phospho-p70^S6K (1:500 dilution), and HSC70 (1:2000 dilution).

FIGURE 7.

Increased skeletal muscle mass in myocilin-expressing transgenic mice compared with wild-type mice. A, increased skeletal muscle mass in legs of transgenic (Tg) mice compared with that of control mice. B, increased weight of dissected gastrocnemius muscles in transgenic mice. C, H&E and Masson's trichrome staining of TA muscle cross-sections (10 μm). Scale bar, 100 μm. D, laminin immunofluorescence of frozen cross-sections (10 μm) of TA muscles from 8-month-old wild-type and transgenic littermates; scale bar, 100 μm. E, cross-sectional area of muscle fibers stained with laminin in D was measured using ImageJ (National Institutes of Health). Size distribution of fibers was plotted as a percentage of total fiber number. F, average fiber size was determined from five independent pairs of mice according to the same analysis as in E. Error bars, S.E. **, p < 0.01.