Gene transfer establishes primacy of striated vs. smooth muscle sarcoglycan complex in limb-girdle muscular dystrophy

Abstract

Limb-girdle muscular dystrophy types 2E and F are characterized by skeletal muscle weakness and often cardiomyopathy and are due to mutations in the genes encoding beta- and delta-sarcoglycan. We previously demonstrated that loss of sarcoglycans in smooth muscle leads to constrictions of the microvasculature that contributes to the cardiac phenotype. It is unclear how vasculature abnormalities affect skeletal muscle. We injected recombinant beta- or delta-sarcoglycan adenoviruses into skeletal muscles of corresponding null mice. We hypothesized that the adenoviruses would not transduce vascular smooth muscle, and we would only target skeletal muscle. Indeed, sustained expression of intact sarcoglycan-sarcospan complex was noted at the sarcolemma, neuromuscular junction, myotendinous junction, and in peripheral nerve, but not in vascular smooth muscle. Gene transfer of the corresponding deleted sarcoglycan gene preserved sarcolemmal integrity, prevented pathological dystrophy and hypertrophy, and protected against exercised-induced damage. We conclude that vascular dysfunction is not a primary cause of beta- and delta-sarcoglycan-deficient muscular dystrophy. In addition, we show successful functional rescue of entire muscles after adenovirus-mediated gene delivery. Thus, virus-mediated gene transfer of sarcoglycans to skeletal muscle in combination with pharmacological prevention of cardiomyopathy constitute promising therapeutic strategies for limb-girdle muscular dystrophies.

Fig. 1.

Restoration of the sarcoglycan–sarcospan complex and nNOS at the sarcolemma upon adenovirus-mediated expression of β- and δ-SG. (a) Quadriceps muscle cryosections from WT, Sgcb-null, Sgcb-null mice injected with recombinant β-SG adenovirus, Sgcd-null, and Sgcd-null mice injected with recombinant δ-SG adenovirus were stained with Abs against α-SG, β-SG, γ-SG, δ-SG, sarcospan (SSPN), and nNOS. (b) Glycoprotein preparations from quadriceps muscle of WT, Sgcd-null, and Sgcd-null mice injected with δ-SG adenovirus were analyzed after 16 days by SDS/PAGE and immunoblotting by using Abs against α-SG, β-SG, γ-SG, δ-SG, and sarcospan (SSPN) as indicated. Abs against α2-dihydropyridine receptor (DHPR) demonstrate equal loading.

Fig. 2.

Sarcoglycan expression is restored at the NMJ, MTJ, and Schwann cells, but not vasculature, on β- and δ-SG gene transfer. (a) Sections from skeletal muscle containing NMJ of WT, Sgcb-null, Sgcb-null mice injected with recombinant β-SG adenovirus, Sgcd-null, and Sgcd-null mice injected with recombinant δ-SG adenovirus were doubly stained with α-SG Ab (α-SG, red) and fluorescein α-bungarotoxin (α-BTX, green). (b) Sections from skeletal muscle of WT, Sgcb-null, Sgcb-null mice injected with recombinant β-SG adenovirus, Sgcd-null, and Sgcd-null mice injected with recombinant δ-SG adenovirus were stained with β-SG Abs. Arrows indicate MTJs. (c) Sections of skeletal muscle containing peripheral nerve of WT, Sgcb-null, Sgcb-null mice injected with recombinant β-SG adenovirus, Sgcd-null, and Sgcd-null mice injected with recombinant δ-SG adenovirus were stained with Abs against β-SG. (d) Sections from skeletal muscle containing smooth muscle of WT, Sgcb-null, and Sgcd-null mice injected with the corresponding sarcoglycan adenovirus were doubly stained with β-SG (β-SG, red) or δ-SG (δ-SG, red) and smooth muscle actin (actin, green) Abs.

Fig. 3.

Adenovirus-mediated gene transfer of δ-SG prevents uptake of gadolinium in tibialis anterior muscle. Contrast agent-enhanced MRI of WT (Top), noninjected (Middle), and right-leg-injected Sgcd-null (Bottom) mice. Cross section images through the left (L) and right (R) tibialis anterior muscles were taken 20 min after injection of gadolinium. T denotes the tail of the mouse. Note lack of uptake of contrast agent in WT muscles; dramatic uptake in Sgcd-null muscles (white areas) and major decrease in enhancement levels in right-side-injected muscles.

Fig. 4.

Adenovirus-mediated expression of β- and δ-SG protects the tibialis anterior muscle from dystrophic damage. (a Left) Severe dystrophic changes including central nucleation, necrosis (horizontal arrows), and dystrophic calcification (vertical arrows) were detected by hematoxylin and eosin staining of cryosections of noninjected tibialis anterior muscles of Sgcb-null and Sgcd-null mice. (Center) Contralateral tibialis anterior muscles 1 mo after i.m. injections of β- or δ-SG adenoviruses. Note that transduced fibers were protected from dystrophic damage. (Right) Serial sections of injected muscles stained with a polyclonal Ab against α-SG. (b) Higher magnification of muscles in a.

Fig. 5.

Injection of δ-SG adenovirus protects against exercised-induced injury. Composite cross-sectional images of tibialis anterior, hamstring, quadriceps, and calf muscles from Sgcd-null mice, exercised Sgcd-null mice, Sgcd-null mice injected with δ-SG adenovirus (Sgcd-null Ad), and exercised Sgcd-null mice injected with δ-SG adenovirus. Staining in green denotes β-dystroglycan (first four columns) or α-SG (last column), and staining in red denotes EBD uptake. (Bars = 220 μm.)

Fig. 6.

Adenovirus-mediated expression of δ- and β-SG protects myofibers from central nucleation and increased muscle mass. (a) Percentage of transduced fibers with centrally located nuclei 2 mo after adenovirus injections into Sgcd-null tibialis anterior (□), hamstring , quadriceps , and calf (▪) muscles. *, Difference from Sgcd-null, P ≈ 0; #, difference from Sgcd-null, P < 0.0015. (b) Tibialis anterior (□) and hamstring (▪) muscle masses of 30-wk-old mice. *, Difference from Sgcb-null, P < 0.026; #, difference from Sgcd-null, P < 0.0002; †, difference from Sgcb-null and WT, P < 0.001 and P < 0.008, respectively; ‡, difference from Sgcd-null, P < 0.027. Statistical significance was examined by using Student's t test.