Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice

Abstract

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:beta1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcbeta1,4[NeuAc(orGc)alpha2, 3]Galbeta1,4GlcNAcbeta-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.

Figures

Fig. 1.

Grip strength measurements in GalNAc transferase (Galgt2) transgenic and nontransgenic wild-type (WT) and mdx mice. Hindlimb and forelimb grip strength was measured in WT, Galgt2 transgenic WT (Galgt2WT), mdx, and Galgt2 transgenic mdx (Galgt2mdx) mice at 2 mo (2mo; A) and 5 mo (5mo; B) of age. Errors are SE for n = 7 (WT 2mo, male), 12 (WT 2mo, female), 18 (Galgt2WT 2mo, male), 10 (Galgt2WT, female), 10 (mdx 2mo, male), 17 (mdx 2mo, female), 8 (Galgt2mdx 2mo, male), 15 (Galgt2mdx 2mo, female), 4 (WT 5mo, male), 8 (WT 5mo, female), 6 (Galgt2WT 5mo, male), 9 (Galgt2WT 5mo, female), 4 (mdx 5mo, male), 8 (mdx 5mo, female), 4 (Galgt2mdx 5mo, male), or 8 (Galgt2mdx 5mo, female) animals. In otherwise identical mice (strain, treatment, or age): *significant difference (P < 0.05) based on age compared with younger mice; #significant difference (P < 0.05) based on WT vs. mdx; $significant difference (P < 0.05) based on Galgt2 overexpression.

Fig. 2.

Galgt2 transgenic WT and mdx muscles are protected from loss of force during eccentric contractions. A: 5-mo-old WT, Galgt2WT, mdx, and Galgt2mdx muscles were compared for force drop during repetitive eccentric contractions. Galgt2WT and Galgt2mdx were both significantly changed relative to either WT or mdx. B: 2-mo-old and 5-mo-old Galgt2WT and Galgt2mdx muscles were compared for force drop during eccentric contractions. C: 2-mo-old Galgt2WT and Galgt2mdx muscles were compared with 3-wk-old (3wk) WT and mdx muscles. The 3-wk-old WT and mdx muscles are as small or smaller in cross-sectional area than 2-mo-old Galgt2WT and Galgt2mdx muscles. P < 0.001 for Galgt2WT 5mo or Galgt2mdx 5mo compared with either WT 5mo or mdx 5mo (in A) and for Galgt2WT 2mo or Galgt2mdx 2mo compared with either WT 3wk or mdx 3wk (in C). Errors are SE for n = 7 (WT 5mo), 8 (Galgt2WT 5mo), 4 (mdx 5mo), 5 (Galgt2mdx 5mo), 3 (Galgt2WT 2mo), 10 (Galgt2mdx 2mo), 2 (WT 3wk), and 6 (mdx 3wk) muscles.

Fig. 3.

Galgt2 overexpression protects myofibers from membrane damage. A: extensor digitorum longus (EDL) muscles from WT, Galgt2WT, mdx, and Galgt2mdx mice were incubated with procion orange after eccentric contraction paradigm, and the percentage of myofibers taking up dye was measured. Muscles were from animals at 5 mo or 3 wk of age. Errors are SE for n = 2 (WT 3wk), 7 (WT 5mo), 8 (Galgt2WT 5mo), 4 (mdx 5mo), 6 (mdx 3wk), or 5 (Galgt2mdx 5mo) muscles. *,#Significant difference (P < 0.05) vs. WT 3 wk and Galgt2WT 5 mo or between mdx 3wk and Galgt2mdx 5 mo. B: correlation between loss of tetanic force after 5 contractions and procion orange uptake. Errors are SE for n as in A.

Fig. 4.

Postnatal Galgt2 overexpression protects mdx muscle from loss of force during eccentric contractions. A: cytotoxic T cell (CT) carbohydrate immunostaining (with the CT2 monoclonal antibody) in the EDL and tibialis anterior (TA) muscle shows overexpression of the CT carbohydrate in a subset of myofibers resulting from Galgt2 transgene overexpression after serotype 8-like recombinant adeno-associated virus (AAV) vector (rAAV8)-CMV-Galgt2(mouse) treatment via the femoral artery 6 wk postinfection. Mock-treated muscle was stained in an identical manner, and exposures are time matched. CT2 staining of large blood vessels occurs normally in these sections. Bar is 200 μm. B: quantification of the average percentage of myofibers overexpressing Galgt2 in rAAV8-CMV-Galgt2-infected mdx muscles. C: mdx muscles infected (via the femoral artery) with 1 × 1012 vg of rAAV8-CMV-Galgt2(mouse) were compared with mock-infected contralateral mdx EDL muscles for force drop during repetitive eccentric contractions. rAAV8-CMV-Galgt2 treatment (green) significantly protected against loss of force compared with mock-treated (contralateral) muscles (blue) (P < 0.001). D: measurement of normalized specific force in rAAV8-CMV-Galgt2-infected and mock-infected contralateral mdx EDL muscles show no significant change. Errors are SE for n = 6 muscles per condition.

Fig. 5.

rAAV8-muscle creatine kinase (MCK)-Galgt2-treated mdx muscles are protected from loss of force during eccentric contractions and have increased normalized specific force equivalent to rAAV8-MCK-microdystrophin-treated mdx muscles. A: mdx muscles infected with 1 × 1011 vg of rAAV8-MCK-Galgt2(human) (green) or with 1 × 1011 vg of rAAV8-MCK-Micro-dys(human) (red) were compared with mock-infected contralateral mdx EDL muscles (blue) and WT (WT C57Bl/10) EDL muscles (black) for force drop during repetitive eccentric contractions at 12 wk postinfection. Both rAAV8-MCK-Galgt2 (Galgt2) and rAAV8-MCK-microdystrophin (Micro-dys) treatment significantly protected against loss of force compared with mock-treated mdx muscles (P < 0.001 for either vs. mdx). B: treatment with either rAAV8-MCK-Galgt2 or rAAV8-MCK-Micro-dys significantly increased normalized specific force relative to mock-treated mdx muscles (P < 0.05 for either vs. mdx). Errors are SE for n = 10 (rAAV8-MCK-microdystrophin, Micro-dys), 12 (rAAV8-MCK-Galgt2, Galgt2), 21 (WT, C57Bl/10), or 25 (mdx) muscles per condition. C: CT carbohydrate immunostaining in the EDL and TA shows overexpression of the CT carbohydrate in a subset of mdx myofibers resulting from Galgt2 transgene overexpression after rAAV8-MCK-Galgt2(human) treatment via the femoral artery. Mock-infected muscle was stained in an identical manner and exposures are time matched. Dystrophin protein immunostaining in the EDL and TA shows overexpression in a subset of mdx myofibers resulting from microdystrophin overexpression after rAAV8-MCK-Micro-dys(human) treatment via the femoral artery. Mock-infected muscle was stained in an identical manner and exposures are time matched. Bar is 200 μm. D: quantification of the average percentage of myofibers overexpressing Galgt2 in rAAV8-MCK-Galgt2(human)- or rAAV8-MCK-Micro-dys(human)-infected muscles. Microdystrophin was expressed in significantly more myofibers than Galgt2 (P < 0.05).

Fig. 6.

Fiber type composition of EDL muscles in all experimental conditions. A: fiber type composition of the EDL muscle of WT, Galgt2WT, mdx, or Galgt2mdx 2-mo-old muscles was determined using ATPase immunohistochemical staining at pH 4.2 to define slow (type 1) fibers, which are black, and fast (type II) subtypes of myofibers, which are not stained. Blood vessels and capillaries are also stained black with this staining method. B: quantitation of fiber type composition showed no change between the four groups. C: fiber type composition of mdx muscles treated with rAAV8-CMV-Galgt2(mouse), rAAV8-MCK-Galgt2(human), rAAV8-MCK-microdystrophin(human), or appropriate mock-infected controls. D: quantitation of fiber type composition showed no change between the five groups. Bar is 200 μm. Errors are SE for n = 6 muscles per condition.