Restoration of muscle functionality by genetic suppression of glycogen synthesis in a murine model of Pompe disease

Abstract

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.

Figure 1.

Prevention of glycogen accumulation in the heart and skeletal muscle of GAA-KO mice by glycogen synthesis inhibition. Glycogen accumulation in cardiac (A) and skeletal muscle (gastrocnemius) (B) was measured in 6-month-old mice. Results are expressed as the mean ± SEM (n ≥ 4). *P < 0.05 compared with WT mice and, #P < 0.05 compared with GAA/GYS1-KO mice. High resolution light microscopy (HRLM) sections of cardiac and skeletal muscle glycogen are presented in (C and D), respectively (PAS, periodic acid Schiff and Richardson's stain, ×600).

Figure 2.

Prevention of cardiomegaly and skeletal muscle atrophy in GAA-KO mice by glycogen synthesis inhibition. The weight (% of body mass) of the heart [1-day-old in (A) and adult in (B)] and gastrocnemius muscle (6-month-old) (C) was measured. The weights are expressed as the mean ± SEM (n ≥ 4). *P < 0.05 compared with WT mice and #P < 0.05 compared with GAA/GYS1-KO mice.

Figure 3.

Staining for LAMP-1 (late endosomes/lysosomes) and LC3 (autophagosomes) in type II myofibers from 6-month-old mice. The endosomal/lysosomal compartment (LAMP-1-positive structures in red) is enlarged in GAA-KO mice (B, D) when compared with WT (A) and GAA/GYS1-KO (C) mice. The autophagosomes (LC3-positive structures in green) are present in the core of the myofiber from GAA-KO (B), but not in WT (A) or in GAA/GYS1-KO mice (C).

Figure 4.

Dystrophin staining of the gastrocnemius and EDL in 3-month-old mice of different genotypes. Gastrocnemius (A–C) and EDL (extensor digitorum longus) (D–I). WT (A, D), GAA-KO (B, E, G, H, I) and GAA/GYS1-KO mice (C, F).

Figure 5.

Quantitative analysis of myofiber composition in the gastrocnemius and soleus muscle from 3-month-old mice. Myofiber areas of the gastrocnemius (A, C) and soleus (B, D) were calculated after dystrophin staining. Results are expressed as the mean ± SEM (n ≥ 3). For each mouse, fiber area or percentage of fibers were measured on more than 500 fibers for the soleus and more than 2000 fibers for the gastrocnemius. The myofiber composition (E and F) was calculated after specific staining with antibodies against type I, IIA, IIB and IIX myosin. Values are expressed as the mean ± SEM (n ≥ 3). *P < 0.05 compared with WT mice and #P < 0.05 compared with GAA/GYS1-KO mice.

Figure 6.

Blood glucose level, glucose and insulin tolerance of 3-month-old mice on a standard diet. Blood glucose level was measured after normal diet (H0), 3 h (H3) or 12 h (H12) of fasting (A). Glucose tolerance test (GTT) (B). Insulin tolerance test (ITT) (C). Values are expressed as the mean ± SEM (n ≥ 3). *P < 0.05 compared with WT mice and #P < 0.05 compared with GAA/GYS1-KO mice.

Figure 7.

Expression of GLUT4 and GLUT1 glucose transporters. Protein levels were analyzed by western blot on plasma membranes (30 μg) isolated from tibialis anterior muscles of mice challenged with 0.2 IU/kg body weight insulin for 5 min. Loading was normalized using anti-M-cadherin antibody (A). Quantification of three independent experiments (B). Data are expressed as the mean ± SEM (n ≥ 4). *P < 0.05 compared with WT mice and #P < 0.05 compared with GAA/GYS1-KO mice. NS: non-significant difference compared with WT in the presence of insulin.

Figure 8.

Muscle function in 11-month-old mice. Maximal tetanic force (A) and fatigue resistance (FR) were tested on the soleus. Results are expressed as the mean ± SEM (n ≥ 12). *P < 0.05 compared with WT mice and #P < 0.05 compared with GAA/GYS1-KO mice.